Combinatorial therapies for the treatment of neoplasias using the opioid growth factor receptor

ABSTRACT

The present invention relates to pharmaceutical compositions for treating neoplasias in an animal or human comprised of a carrier and therapeutically effective amounts of at least one chemotherapeutic agent along with the biotherapeutic endogenous pentapeptide Met-enkephalin, referred to as opioid growth factor. Also provided are methods of treating neoplasias in an animal or human in need of such treatment, comprising the administration to the animal or human therapeutically effective amounts of a pharmaceutical composition comprised of a carrier and therapeutically effective amounts of at least one neoplasia-treating agent, such as a chemotherapeutic agent or radiation, along with opioid growth factor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 of a provisionalapplication Ser. No. 60/548,021 filed Feb. 26, 2004, which applicationis hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to therapeutic formulations for use inthe treatment of neoplasias. More specifically, the invention relates topharmaceutical formulations comprised of chemotherapeutic agents andbiotherapeutic agents for treating neoplasias. Methods for treatingneoplasias by administering combinatorial formulations ofneoplasia-treating agents, such as chemotherapeutic and/or radiation,along with biotherapeutic agents are also disclosed.

DESCRIPTION OF RELATED ART

Cancer is the second leading cause of death in the United States,surpassed only by heart disease. According to the American CancerSociety, approximately 556,000 Americans die from cancer each year-anaverage of more than 1,500 cancer deaths each day (Jemal, A. et al., CACancer J. Clin., 55, 10-30, 2005). Of the different cancers notincluding the skin cancers, lung cancer is the leading cause of cancerdeath for both men and women; breast cancer is the second leading causeof cancer death in women; prostate cancer is the second leading cause ofcancer death in men and colorectal cancer is the third most frequentlydiagnosed form of cancer.

Pancreatic cancer is the most lethal human cancer with median survivalfor all stages of pancreatic cancer being less than 3-5 months fromdiagnosis. (CA Cancer J. Clin, 2004 54:8-20). The five-year survivalrate is 3% or less. In spite of treatment efforts of surgery, radiation,and chemotherapy, the survival rate remains unchanged. (CA Cancer J.Clin, 2004) The incidence of pancreatic cancer is only 0.01% in theUnited States, but it is associated with the deaths of over 30,000individuals each year, making this the most common in terms of cancermortality. (Jemal, A. et al., CA Cancer J. Clin., 55, 10-30, 2005).Approximately 85-90% of symptomatic patients have advanced disease as aresult of local infiltration or metastases at the time of diagnosis, andthe prognosis for these individuals is extremely poor. (CA Cancer J.Clin 2005). Although some advances in treatment have been made thatinclude surgery, chemotherapy, radiation therapy, immunotherapy, andhormonal therapy, pancreatic cancer remains a profound challenge interms of prevention, diagnosis, prognosis and therapy.

At the time of diagnosis, up to around to around about 20% of pancreatictumors can be removed by surgery. (Lancet 2004; 363:1049-57). When thetumor is confined to the pancreas but cannot be removed, a combinationof radiotherapy and chemotherapy is usually performed. When the tumorhas metastasized to other organs, such as the liver, chemotherapy aloneis usually used. The standard chemotherapy agent is gemcitabine, butother drugs may be used. Gemcitabine essentially provides onlypalliative improvement in patients.

Head and neck cancer is the sixth ranking cancer in the world, and thethird most common neoplasia in developing nations. In the United States,the incidence of cancer of the aerodigestive tract accounts forapproximately 40,000 new cases each year, with over 11,000 fatalitiesrecorded annually (Jemal, A. et al., CA Cancer J. Clin., 55, 10-30,2005). More than 90% of head and neck cancers are squamous cellcarcinomas (SCCHN), with the oral cavity and pharynx being the mostcommon sites for SCCHN, followed by the larynx. Surgery, radiotherapyand chemotherapy, and combinations thereof, are all considered fortreatment. Unfortunately, there is over a 50% chance of recurrence ofSCCHN within two years, and the five-year survival is approximately 50%for all sites and stages. Moreover, in the last twenty-five years, thefive-year survival of patients with SCCHN has not changed appreciably(Jemal, A. et al., CA Cancer J. Clin., 55, 10-30, 2005).

Peptide growth factors and their receptors have been implicated in SCCHNand pancreatic cancer, as well as in a number of other cancers(Sugerman, P. B. et al., Oral Dis., 1, 172-188, 1995). Some of thepeptides found to be expressed in pancreatic cancer and SCCHN includeepidermal growth factor (EGF), transforming growth factors α and β,basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF),platelet derived growth factor (PDGF), and keratinocyte growth factor(KGF).

One group of peptides, the endogenous opioids, are believed to beimportant in the growth of normal, neoplastic, renewing and healingtissues, as well as in prokaryotes and eukaryotes (Zagon, I. S. et al.,In: Cytokines: Stress and Immunity. Plotnikoff NP et al., (eds). CRCPress, Boca Raton, Fla., pp. 245-260, 1999). Met-enkephalin, anendogenous opioid peptide, is directly involved in growth processes, andserves as a negative regulator in a wide variety of cells and tissues(Zagon, I. S. et al., In: Receptors in the Developing Nervous System.Vol. 1. Zagon, I. S. and McLaughlin, P. J. (eds). Chapman and Hall,London, pp. 39-62, 1993). In view of its function (growth) anddistribution (neural and non-neural), the peptide has been termed opioidgrowth factor (OGF).

Cancer chemotherapeutic agents are used for their lethal action tocancer cells. Unfortunately, few such drugs differentiate between acancer cell and other proliferating cells. Chemotherapy generallyrequires use of several agents concurrently or in planned sequence.Combining more than one agent in a chemotherapeutic treatment protocolallows for: (1) the largest possible dose of drugs; (2) drugs that workby different mechanisms; (3) drugs having different toxicities; and (4)the reduced development of resistance.

Chemotherapeutic agents mainly affect cells that are undergoing divisionor DNA synthesis, thus slow growing malignant cells, such as lung canceror colorectal cancer, are often unresponsive. Furthermore, mostchemotherapeutic agents have a narrow therapeutic index. Common adverseeffects of chemotherapy include vomiting, stomatitis, and alopecia.Toxicity of the chemotherapeutic agents is often the result of theireffect on rapidly proliferating cells, which are vulnerable to the toxiceffects of the agents, such as bone marrow or from cells harbored fromdetection (immunosuppression), gastrointestinal tract (mucosalulceration), skin and hair (dermatitis and alopecia).

Many potent cytotoxic agents act at specific phases of the cell cycle(cell cycle dependent) and have activity only against cells in theprocess of division, thus acting specifically on processes such as DNAsynthesis, transcription, or mitotic spindle function. Other agents arecell cycle independent. Susceptibility to cytotoxic treatment,therefore, may vary at different stages of the cell life cycle, withonly those cells in a specific phase of the cell cycle being killed.Because of this cell cycle specificity, treatment with cytotoxic agentsneeds to be prolonged or repeated in order to allow cells to enter thesensitive phase. Non-cell-cycle-specific agents may act at any stage ofthe cell cycle; however, the cytotoxic effects are still dependent oncell proliferation. Cytotoxic agents thus kill a fixed fraction of tumorcells, the fraction being proportionate to the dose of the drugtreatment.

Numerous neoplasia-treating agents are currently in use today, includingany chemotherapeutic agents, and biotherapeutic agents as well asradiation therapy. There are numerous types of chemotherapeutic agents,including alkylating agents, nitrosoureas, antimetabolites, antitumorantibiotics, mitotic inhibitors, corticosteroid hormones, sex hormones,immunotherapy or others such as L-asparaginase and tretinoin. Some arebriefly discussed below.

A widely used current chemotherapeutic agent is gemcitabine. Gemcitabineis a pyrimidine analogue that belongs to a general group of chemotherapydrugs known as antimetabolites and that also acts as aradiation-sensitizing agent. Gemcitabine exhibits cell phasespecificity, primarily killing cells undergoing DNA synthesis, i.e., theS-phase, and also blocks the progression of cells through the G₁/S-phaseboundary.

Gemcitabine is an approved chemotherapeutic agent for a wide range oftumors that include, but are not limited to, pancreatic and colorectalcarcinoma. The efficacy of gemcitabine is marginal, however, and lifeexpectancy is rarely extended, particularly for pancreatic cancerpatients. Side effects of gemcitabine administration are relatively mildwhen compared to other chemotherapeutic agents, consisting ofmyelosuppression with increased risk of infection, decreased plateletcount with increased risk of bleeding, nausea, vomiting, increased liverfunction blood tests and fatigue. Gemcitabine, in general, however, hasreplaced other therapies because of its less toxic effects on thepatient, and hence, a better quality of life.

The platin family of chemotherapeutics consists primarily of cisplatinand carboplatin. Cisplatin is an inorganic platinum complex thatdisrupts the DNA helix by forming intra- and interstrand cross-links.Cisplatin also reacts, however, with nucleophils of other tissues,causing toxic effects on the kidney and on the eight cranial nerve(which is responsible for causing intense nausea and vomiting). Otherside effects include renal toxicity, ototoxicity manifested by tinnitusand hearing loss, and mild to moderate myelosuppression. Carboplatindiffers from cisplatin mainly with respect to side effects.Myelosuppression is the dose-limiting toxicity for carboplatin with verylittle of the renal, neurologic, or ototoxicities that are encounteredwith cisplatin.

Paclitaxel is a natural, although quite toxic, substance derived fromthe yew tree that is chemically altered to produce a powerful antimicrotubule chemotherapeutic agent indicated for the treatment ofmetastatic breast cancer, metastatic ovarian cancer, and Kaposi'ssarcoma. Paclitaxel also has been used to treat SCCHN, non-small celllung cancer, small cell lung cancer and bladder cancer. Side effectscommonly encountered with paclitaxel administration include nausea andvomiting, loss of appetite, change in taste, thinned or brittle hair,pain in the joints of the arms or legs lasting 2-3 days, changes in thecolor of nails and tingling in hands or toes.

The chemotherapeutic agent, 5 fluorouracil (5-FU), has been one of themajor antimetabolites used in a variety of solid cancers since the1960s. 5-FU prevents cells from making DNA and RNA by interfering withthe synthesis of nucleic acids, thus disrupting the growth of cancercells. 5-FU is used alone or in combination in the adjuvant treatment ofbreast, colon, gastrointestinal and head or neck cancer. 5-FU also isused as a palliative therapy of inoperable malignant neoplasms, such asof the gastrointestinal tract, breast, liver, genitourinary system andpancreas. 5-FU has many common side effects, including myelosuppressionwith increased risk of infection and bleeding, darkening of skin andnail beds, nausea, vomiting, sores in mouth or on the lips, thinninghair, diarrhea, brittle nails, increased sensitivity to the sun and dry,flaky skin.

There exists a need, therefore, for a therapeutic formulation to treatvarious types of cancer and, in particular, pancreatic cancer and SCCHN,which demonstrates enhanced efficacy and survival rates with reducedconcomitant side effects and toxicity commonly encountered withchemotherapeutic agents.

SUMMARY OF THE INVENTION

The present invention provides for the first time a carcinotherapeuticpharmaceutical composition and/or treatment method for treatingneoplasias in an animal or human comprised of a carrier andtherapeutically effective amounts of at least one neoplasia treatingagent, such as chemotherapeutic agent or radiation therapy (agent) andthe biotherapeutic endogenous pentapeptide Met-enkephalin, referred toas opioid growth factor (OGF). As used herein, a carcinotherapeuticcomposition refers to a composition that includes both chemotherapeuticand biotherapeutic agents for the treatment of all neoplasias, includingbut not limited to true carcinomas but also other cancers such assarcomas, melanomas, etc.

As used herein the term “OGF or Met-enkephalin” shall be interpreted toinclude all modifications, substitutions, truncations or derivatives ofOGF or Met-enkephalin which retain the ability to interact with the OGFreceptor in a similar fashion to OGF as described herein. This alsoincludes synthetic or any other compound which mimics the biologicalactivity of OGF in its interaction with the OGF receptor.

The present invention also provides a method of treating neoplasias inan animal or human in need of such treatment, comprising theadministration to the animal or human therapeutically effective amountsof each of at least one neoplasia-treating agent and OGF. A wide varietyof neoplasia-treating agents have been shown to be effective when usedin combination with OGF including anti-metabolites, cytosine analogs,cross linking agents and the like. The effects of OGF are mediatedthrough the OGFr and thus it is postulated that any chemotherapeuticagent, or biotherapeutic agent, will have similar effects, includingradiation therapy. Some examples of chemotherapeutic agents that can beused in accordance with the invention include without limitation,Neoplasia-treating agents can include any chemotherapeutic agents aswell as radiation therapy. There are numerous types of chemotherapeuticagents, any of which may be used according to the invention, include butare not limited to alkylating agents, nitrosoureas, antimetabolites,antitumor antibiotics, mitotic inhibitors, corticosteroid hormones, sexhormones, immunotherapy or others such as L-asparaginase and tretinoin.

The combination of the biotherapeutic OGF and neoplasia treating agentis in most cases at least additive which will allow for a reduction intoxicity of the treatment as a similar result may be achieved with alower dose of the neoplasia treating agent. This is important as many ofthese agents are highly toxic and should be used in as small dose aspossible. In at least one protocol the reduction in toxicity was seen inaddition to the additive nature of the agents. Often the result of thecombination is a synergistic effect, i.e. the reduction in cells isgreater than the sum of each of the agents alone. The effects of the OGFare blocked by naloxone indicating that the OGF effect is entirelymediated by the OGFr.

In yet another embodiment the OGFr may be introduced to tumor cells in asuicide type treatment protocol where tumor or neoplasia cells will besensitized to the anti-neoplastic treatment by the introduction ofadditional OGFr receptors to the cells so that OGF may interact with asmany cells as possible in mediating and potentiating the effect of thetherapy.

Neoplasias that can be treated according to the method of the presentinvention include any neoplasia cell that has an OGFr, this can includewithout limitation, pancreatic cancer, squamous cell cancer of the headand neck, breast cancer, colorectal cancer, renal cancer, brain cancer,prostate cancer, bladder cancer, bone or joint cancer, uterine cancer,cervical cancer, endometrial cancer, multiple myeloma, Hodgkin'sdisease, non-Hodgkin's lymphoma, melanoma, leukemias, lung cancer,ovarian cancer, gastrointestinal cancer, Kaposi's sarcoma, liver cancer,pharyngeal cancer or laryngeal cancer.

The effective therapeutic amount of OGF that can be administeredaccording to the composition in an intravenous protocol for examplebetween about 20 to 1000 μg/kg body weight per day, preferably about 100to 400 μg/kg body weight. OGF may be administered at least once a week,and as frequently as multiple times daily, throughout the entiretreatment period depending on the route of administration. OGF isnon-toxic and may be administered in accordance with essentially anyeffective dose. The mode of administration, i.e. intravenous,subcutaneous, etc. may also alter the effective dose and timetable ofdrug administration, but such can be determined through routineexperimentation. The antineoplastic agent may be administeredsequentially, or simultaneously with the administration of OGF, at leastone neoplasia treating agent is administered to an animal or human intherapeutically effective amounts of, for example, between about 20 to3000 mg/m², preferably about 100 to 1000 mg/m², over a period of betweenabout 10 to 60 minutes, and preferably about 30 minutes, at least once aweek for about three to ten weeks, preferably seven weeks. After one tothree weeks, preferably one week, of rest, the chemotherapeutic agent isadministered over a period of between about 10 to 60 minutes, preferablyabout 30 minutes, for about one to five weeks, preferably three weeks.Administration of the chemotherapeutic agent can repeat every two toeight weeks, preferably four weeks, in the absence of diseaseprogression or unacceptable toxicity. Subcutaneous or implant deliverywill also be effective.

In another embodiment of the present invention, OGF is administered inan effective dose of about 20 to 1000 μg/kg body weight, preferablyabout 100 to 400 μg/kg body weight at least three times a week,preferably daily, during the course of radiation therapy.

In yet another embodiment of the present invention, OGF is administeredin an effective dose of about 20 to 1000 μg/kg body weight, preferablyabout 100 to 400 μg/kg body weight at least three times a week,preferably daily, with chemotherapy during the course of radiationtherapy.

The route of administration of the antineoplastic agent(s) and opioidgrowth factor includes, without limitation, parenteral administration,namely intravenous, intramuscular or intraperitoneal, subcutaneous,implanted osmotic pump or transdermal patch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph representing a 96-hour growth curve for SCC-1 cellsbeing treated with paclitaxel (Taxol) and/or OGF. Each data pointrepresents the average absorbency for 10 wells±S.E.M. Significancevalues for each timepoint can be found on Table 1.

FIG. 2 is a graph representing a 96-hour growth curve for SCC-1 cellsbeing supplemented with carboplatin and/or OGF. Each data pointrepresents the average absorbency for 10 wells±S.E.M. Significancevalues for each timepoint can be found on Table 2.

FIG. 3 shows the growth of SCC-1 SCCHN cells in athymic nude mice.Timepoint 1 signifies the first day that tumors became measurable ineach treatment group. Tumor volumes were recorded every day and averagesfrom 2 consecutive days represent the timepoints on the x-axis.

FIG. 4 shows the final termination weights for athymic nude miceinoculated with SCC-1 SCCHN cells. Bars represent the mean values forweight for the entire treatment group at the time of termination (Day50). Significant from controls at p<001 (***), significant from OGF atp<0.001 (+++), and significant from Taxol/OGF at p<0.001 ({circumflexover ( )}{circumflex over ( )}{circumflex over ( )}).

FIG. 5 shows a survival curve representing the percent of surviving micein each of the four groups over the course of the 50-day study.

FIG. 6 is a graph representing a 96-hour growth curve for MiaPaCa-2cells treated with gemcitabine and/or OGF. Each data point representsthe average absorbency for 10 wells±S.E.M. Significance values for eachtimepoint can be found on Table 3.

FIG. 7 is a graph representing a 96-hour growth curve for MiaPaCa-2cells being treated with 5-FU and/or OGF. Each data point represents theaverage absorbency for 10 wells±S.E.M. Significance values for eachtimepoint can be found on Table 4.

FIG. 8 shows the growth of MiaPaCa-2 human pancreatic cancer cells inathymic nude mice. Timepoint 1 signifies the first day that tumorsbecame measurable in each treatment group. Tumor volumes were recordedevery day and averages from 2 consecutive days represent the timepointson the x-axis. Graph is meant to show growth trends once tumors becamemeasurable in each group. Graph disregards latency to measurable tumordevelopment to illustrate this trend.

FIG. 9 shows the growth of MiaPaCa-2 human pancreatic cancer cellsagainst time subjected to daily addition of the above drug regiments.Values represent the means from 4 wells/timepoint±S.E.M. Significancevalues can be found on Table 5.

FIG. 10 shows cell proliferation assays of MIA PaCa-2 cells subjected toOGF (10⁻⁶ M) and/or gemcitabine (10⁻⁸) (Gemzar) for 96 hr. Drugs or anequivalent volume of sterile water (controls) were added 24 hr (0 hr)after seeding in 6-well plates; media and drugs were replaced daily.Data represent means±SEM for at least 4 wells per treatment at each timepoint. Significantly different from controls at p<0.01 (**) and p<0.001(***). Significantly different from OGF-treated cultures at p<0.001(+++). Significantly different from cultures treated with gemcitabinealone at p<0.001 ({circumflex over ( )}{circumflex over ( )}{circumflexover ( )}).

FIG. 11 depicts growth of MIA PaCa-2 cells treated with 5-FU (10⁻⁶ M)and/or OGF (10⁻⁶ M) as measured by the MTS assay (96-well plates).Values represent mean absorbencies±SEM for 10 wells at each time point.Significantly different from controls at p<0.05 (*), p<0.01 (**), andp<0.001 (***). Significantly different from OGF-treated cultures atp<0.001 (+++). Significantly different from 5-FU-treated cultures atp<0.01 ({circumflex over ( )}{circumflex over ( )}) and p<0.001({circumflex over ( )}{circumflex over ( )}{circumflex over ( )}).

FIG. 12 depicts receptor mediation of the growth inhibitory effects ofgemcitabine and/or OGF in MIA PaCa-2 cells. The number of MIA PaCa-2cells at 96 hr as measured by the MTS assay after being subjected to OGF(10⁻⁶ M), the opioid antagonist naloxone (10⁻⁶ M), gemcitabine (Gemzar)(10⁻⁸ M), or combinations of these compounds; controls were treated withan equivalent volume of sterile water. Compounds and media were replacedevery 24 hr. Data represent mean absorbency±SEM for 10 wells/treatmentat 96 hr. Significantly from controls at p<0.001 (***). NS=notsignificant.

FIG. 13 shows reversibility of the growth inhibitory effects on MIAPaCa-2 cells treated with OGF and/or gemcitabine (Gemzar). Cells wereseeded into 96-well plates and treated with drugs for 48 hr. At 48 hr,half of the plates continued to receive the same drugs for an additional48 hr, and half of the plates were treated with sterile water for 48 hr.Control cultures received sterile water throughout the 96 hr. Compoundsand media were replaced daily. A. Growth of cells in the reversibilityexperiments. B. Cell number at 96 hr in the treatment groups. All datarepresent mean absorbency±SEM for 10 wells/treatment. Comparisonsbetween cell number of cultures maintained with drugs or cultures withdrugs replaced by vehicle (reversal) are presented. NS=not significant.

FIG. 14 shows growth of MIA PaCa-2 cells grown in 96-well plates treatedwith a variety of endogenous and exogenous opioids at a concentration of10⁻⁶ M. Data represent mean absorbency values±SEM for 10wells/treatment. Significantly different from controls at p<0.001 (***).

FIG. 15 shows effects of gemcitabine (10⁻⁸ M) (Gemzar) and/or OGF (10⁻⁶M) on PANC-1 cells grown in 6-well plates. Data represent means±SEM for4 well at 72 hr of treatment. Significantly different from controls atp<0.001 (***), from OGF at p<0.01 (++), and from the respective dosagesof gemcitabine at p<0.001 ({circumflex over ( )}{circumflex over( )}{circumflex over ( )}).

FIG. 16 shows growth of MIA PaCa-2 tumors xenografted into nude mice.Animals were injected with either 10 mg/kg OGF daily, 120 mg/kggemcitabine every 3 days (Gemzar); 10 mg/kg OGF daily and 120 mg/kggemcitabine every 3rd day (Gemzar/OGF), or 0.1 ml of sterile salinedaily (Control). A. Tumor volumes monitored for the 45 days of theexperiment. Values represent means±SEM for all mice in the group (seeResults for statistical comparisons). B. Rates of tumor growth for the45-day experimental period. Tumor volumes were log-transformed andslopes of the lines were calculated. Significantly different fromcontrols at p<0.001(***), from OGF at p<0.001 (+++), and fromgemcitabine at p<0.001 ({circumflex over ( )}{circumflex over( )}{circumflex over ( )}).

FIG. 17 sows growth (cell number determined by a hemacytometer) of SCC-1cells subjected to OGF (10⁻⁶ M) and/or paclitaxel (10⁻⁸ M) (=Taxol) overa 96-hr period. Drugs or an equivalent volume of sterile water (Control)were added 24 hr after seeding 100,000 cells into 6-well plates; mediaand drugs were replaced daily. A. Growth curve data represent means±SEfor at least 4 wells/treatment at each time point. Significantlydifferent from controls at p<0.05 (*), p<0.01 (**), and p<0.001 (***).Significantly different from OGF-treated cultures at p<0.01 (++) andp<0.001 (+++). Significantly different from paclitaxel-treated culturesat p<0.001 ({circumflex over ( )}{circumflex over ( )}{circumflex over( )}). B. Rates of growth calculated from overall slopes of the growthcurves. Data represent the slopes (number of cells/hr) of the curves±SE.Significantly different from controls at p<0.05 (*) and p<0.01 (**).Growth rates for the cells treated with combined therapy also differedfrom OGF-treated cells at p<0.01 (++), and from cells subjected topaclitaxel alone at p<0.05 ({circumflex over ( )}).

FIG. 18 depicts growth of SCC-1 cells treated with carboplatin and/orOGF as measured by the MTS assay. Values represent mean absorbencies±SEfor 10 wells at each time point. Significantly different from controlsat p<0.001 (***). Significantly different from OGF-treated cultures atp<0.001 (+++). Significantly different from carboplatin-treated culturesat p<0.001 ({circumflex over ( )}{circumflex over ( )}{circumflex over( )}).

FIG. 19 shows OGFr mediation of the growth inhibitory effects ofpaclitaxel and/or OGF in SCC-1 cells. The number of SCC-1 cells at 96 hras measured by the MTS assay after being subjected to OGF (10⁻⁶ M), theopioid antagonist naloxone (10⁻⁶ M), paclitaxel (Taxol) (10⁻⁸ M), orcombinations of these compounds; controls were treated with anequivalent volume of sterile water. Compounds and media were replacedevery 24 hr. Data represent mean absorbency±SE for 10 wells/treatment.Significantly different from controls at p<0.001 (***). NS=notsignificant.

FIG. 20 shows the reversibility of the growth inhibitory effects onSCC-1 cells treated with OGF and/or paclitaxel (Taxol). Cells wereseeded into 96-well plates and treated with drugs for 48 hr. At 48 hr,half of the plates continued to receive the same drugs for an additional48 hr, and half of the plates were treated with sterile water for 48 hr.Control cultures received sterile water throughout the 96 hr. Compoundsand media were replaced daily. A. Growth of cells in the reversibilityexperiments. B. Cell number at 96 hr in the treatment groups. All datarepresent mean absorbency±SE for 10 wells/treatment. Comparisons betweencell number of cultures maintained with drugs or the drugs replaced withvehicle (reversal) are presented.

FIG. 21 depicts the growth of SCC-1 cells treated with a variety ofendogenous and exogenous opioids. Data represent mean absorbencyvalues±SE for 10 wells/treatment. Significantly different from controlsat p<0.001 (***).

FIG. 22 shows the evaluation of apoptosis in SCC-1 cells treated withOGF and/or paclitaxel for 24, 72, to 144 hours. Cells were seeded into6-well plates, treated with drugs and, at appropriate times, stainedwith caspase-3. Caspase-3 activity was measured by flow cytometry on10,000 cells/treatment/time. Data represent the percent caspase positivecells (mean±SE) for 3 samples for each treatment at each time point.Significantly different from controls at p<0.001 (***) and from OGF atp<0.001 (+++). Cells exposed to the combined therapy also differed frompaclitaxel treated cells at p<0.001 ({circumflex over ( )}{circumflexover ( )}{circumflex over ( )}).

FIG. 23 shows the evaluation of DNA synthesis by monitoring BrdUincorporation in SCC-1 cells treated with OGF and/or paclitaxel for 24hr or 72 hr. Data represent the percent BrdU positive cells (mean±SE)from analysis of at least 1000 cells for each treatment at each timepoint. Significantly different from controls at p<0.05 (*), p<0.01 (**),and p<0.001 (***), and from the OGF group at p<0.001 (+++).

FIG. 24 shows the effects of paclitaxel and/or OGF on CAL-27 cells, apoorly-differentiated SCCHN cell line. Data represent means±SEM for 4samples at 48 hr of treatment. Significantly different from controls atp<0.001 (***), from OGF at p<0.001 (+++), and from the respectivedosages of paclitaxel at p<0.01 ({circumflex over ( )}{circumflex over( )}).

FIG. 25 shows changes in tumor volume over the 50 days of the experimentanalyzed using a non-linear mixed effects model for clustered data.These analyses were performed to accommodate the marked loss ofpaclitaxel mice beginning on day 20. Tumor volumes of mice in all 3treatment groups were significantly (p<0.001) smaller than controls.Moreover, tumor volumes for mice receiving combined therapy weresignificantly (p<0.001) smaller than tumor sizes in groups receivingeither treatment alone. Animals were given intraperitoneal injections ofeither sterile saline (0.1 ml; Control) daily, OGF (10 mg/kg) daily,paclitaxel (8 mg/kg; Taxol) every other day, or paclitaxel every otherday and OGF daily (Taxol/OGF).

FIG. 26 shows body weights of mice treated with either OGF (10 mg/kg,daily) and/or paclitaxel (8 mg/kg every 2 days; Taxol); control animalsreceived 0.1 ml sterile saline (Control). Body weights were recordedevery 7 days; values represent means±SEM. No significant differences inbody weights between Control, OGF, or Taxol groups were recorded.Significantly different from control group at p<0.05 (*) and p<0.001(***), from the OGF group at p<0.01 (++) and p<0.001 (+++), and from theTaxol/OGF group at p<0.05 ({circumflex over ( )}) and p<0.001({circumflex over ( )}{circumflex over ( )}{circumflex over ( )}).

FIG. 27 shows the survival curves of mice inoculated with 2×10⁶ SCC-1squamous cells of the head and neck and treated with either OGF (10mg/kg, daily) and/or paclitaxel (8 mg/kg every 2 days; Taxol); controlanimals received 0.1 ml sterile saline (Control). Kaplan-Meier curveswere analyzed and the survival of mice receiving only paclitaxel wassignificantly different from all other groups at p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for the first time a carcinotherapeuticpharmaceutical composition and method for treating neoplasias in ananimal or human comprised of a carrier and therapeutically effectiveamounts of at least one chemotherapeutic agent and the biotherapeuticendogenous pentapeptide Met-enkephalin, referred to as opioid growthfactor (OGF).

The present invention also provides a method of treating neoplasias inan animal or human in need of such treatment, comprising theadministration to the animal or human therapeutically effective amountsof each of at least one neoplasia-treating agent and OGF.Neoplasia-treating agents can include any biotherapeutic agents,radiopharmaceuticals, and chemotherapeutic agents as well as radiationtherapy. There are numerous types of chemotherapeutic agents, any ofwhich may be used according to the invention. These include alkylatingagents, nitrosoureas, antimetabolites, antitumor antibiotics, mitoticinhibitors, corticosteroid hormones, sex hormones, immunotherapy orothers such as L-asparaginase and tretinoin. Examples of biotherapeuticagents include but are not limited to interferon, interleukin, tumorderived activated cells. Radionuclides such as Iodine¹²⁵, are alsopertinent as well as radiation therapy from gamma or x-rays.

Chemotherapeutic alkylating agents work directly on DNA to prevent thecancer cell from reproducing. As a class of drugs, these agents are notphase-specific (in other words, they work in all phases of the cellcycle). These drugs are active against chronic leukemias, non-Hodgkin'slymphoma, Hodgkin's disease, multiple myeloma, and certain cancers ofthe lung, breast, and ovary. Examples of alkylating agents includebusulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide,ifosfamide, dacarbazine (DTIC), mechlorethamine (nitrogen mustard), andmelphalan.

Nitrosoureas act in a similar way to alkylating agents. They interferewith enzymes that help repair DNA. These agents are able to travel tothe brain so they are used to treat brain tumors as well asnon-Hodgkin's lymphomas, multiple myeloma, and malignant melanoma.Examples of nitrosoureas include carmustine (BCNU) and lomustine (CCNU).

Antimetabolites are a class of drugs that interfere with DNA and RNAgrowth. These agents work during the S phase and are used to treatchronic leukemias as well as tumors of the breast, ovary, and thegastrointestinal tract. Examples of antimetabolites include5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine(ara-C), and fludarabine.

Antitumor antibiotics interfere with DNA by stopping enzymes and mitosisor altering the membranes that surround cells. (They are not the same asantibiotics used to treat infections.) These agents work in all phasesof the cell cycle. Thus, they are widely used for a variety of cancers.Examples of antitumor antibiotics include dactinomycin, daunorubicin,doxorubicin (Adriamycin), idarubicin, and mitoxantrone.

Mitotic inhibitors are plant alkaloids and other compounds derived fromnatural products. They can inhibit, or stop, mitosis or inhibit enzymesfor making proteins needed for reproduction of the cell. These workduring the M phase of the cell cycle. Examples of mitotic inhibitorsinclude paclitaxel, docetaxel, etoposide (VP-16), vinblastine,vincristine, and vinorelbine.

Steroids are natural hormones and hormone-like drugs that are useful intreating some types of cancer (lymphoma, leukemias, and multiplemyeloma) as well as other illnesses. When these drugs are used to killcancer cells or slow their growth, they are considered chemotherapydrugs. They are often combined with other types of chemotherapy drugs toincrease their effectiveness. Examples include prednisone anddexamethasone.

Sex hormones, or hormone-like drugs, alter the action or production offemale or male hormones. They are used to slow the growth of breast,prostate, and endometrial (lining of the uterus) cancers, which normallygrow in response to hormone levels in the body. These hormones do notwork in the same ways as standard chemotherapy drugs. Examples includeanti-estrogens (tamoxifen, fulvestrant), aromatase inhibitors(anastrozole, letrozole), progestins (megestrol acetate), anti-androgens(bicalutamide, flutamide), and LHRH agonists (leuprolide, goserelin).

Some drugs are given to people with cancer to stimulate their immunesystems to more effectively recognize and attack cancer cells. Thesedrugs offer a unique method of treatment, and are often considered to beseparate from “chemotherapy.”

Some chemotherapy drugs act in slightly different ways and do not fitinto any of the other categories. Examples include such drugs asL-asparaginase and tretinoin.

The combination therapy has been exemplified herein with the alkylatingagent, carboplatin, the antimetabolite 5-FU, and gemcitabine, and amitotic inhibitor Paclitaxel.

Neoplasias that can be treated according to the method of the presentinvention include, without limitation, pancreatic cancer, squamous cellcancer of the head and neck, breast cancer, colorectal cancer, renalcancer, brain cancer, prostate cancer, bladder cancer, bone or jointcancer, uterine cancer, cervical cancer, endometrial cancer, multiplemyeloma, Hodgkin's disease, non-Hodgkin's lymphoma, melanoma, leukemias,lung cancer, ovarian cancer, gastrointestinal cancer, Kaposi's sarcoma,liver cancer, pharyngeal cancer or laryngeal cancer.

The effective therapeutic amount of OGF that can be administeredaccording to the composition and method of the present invention for anintravenous therapy is between about 20 to 1000 μg/kg body weight perday, preferably about 100 to 400 μg/kg body weight per day. OGF may beadministered at least three times a week, and as frequently as oncedaily, throughout the entire treatment period. OGF is safe and nontoxicand may be administered in essentially any amount necessary to beeffective. The route of administration (intravenous, subcutaneous, etc)may affect the amounts than can be given however this is all determinedthorough routine experimentation. Sequentially or simultaneously withthe administration of OGF, at least one chemotherapeutic agent isadministered to an animal or human in therapeutically effective amountsof between about 20 to 3000 mg/m², preferably about 100 to 1000 mg/m²,over a period of between about 10 to 60 minutes, and preferably about 30minutes, at least once a week for about three to ten weeks, preferablyseven weeks. After one to three weeks, preferably one week, of rest, thechemotherapeutic agent is administered over a period of between about10-60 minutes, preferably about 30 minutes, for about one to five weeks,preferably three weeks. Administration of the chemotherapeutic agent canrepeat every two to eight weeks, preferably four weeks, in the absenceof disease progression or unacceptable toxicity.

In another embodiment of the present invention, OGF is administered inan effective dose of about 20 to 1000 μg/kg body weight, preferablyabout 100 to 400 μg/kg body weight at least three times a week,preferably daily, during the course of radiation therapy. The route ofadministration of the chemotherapeutic agent(s) and opioid growth factorinclude, without limitation, parenteral administration, namelyintravenous, intramuscular or intraperitoneal, subcutaneous, implantedslow release osmotic minipump or transdermal patch.

The OGF pentapeptide is a constitutively expressed autocrine inhibitorygrowth factor in a wide variety of cells and tissues both in vivo and invitro, and under normal (e.g., homeostatic development) and abnormal(e.g., cancer, wound healing) conditions. The action of OGF in vitro isstereospecific, reversible, non-cytotoxic, independent of serum andoccurs at physiologically relevant concentrations.

In particular, in a using a human pancreatic cancer cell line, thecombination of OGF and gemcitabine reduced cell number from controllevels by 26% to 46% within 48 hr, and resulted in a growth inhibitiongreater than that of the individual compounds. The combination of OGFand gemcitabine also repressed the growth of a second pancreatic cancercell line. In vivo, addition of OGF to gemcitabine therapy in nude micereduces tumor volume more than either compound alone. Tumor weight andtumor volume were reduced from control levels by 36% to 85% in the OGFand/or gemcitabine groups on day 45 and the group of mice exposed to acombination of OGF and gemcitabine had decreases in tumor size of 62% to77% from the OGF or the gemcitabine alone groups.

OGF in combination with 5-fluorouracil also depressed cell growth morethan either agent alone in a pancreatic cancer cell line.

Similar effects were also observed in squamous cancer cell lines. Thecombination therapy of paclitaxel and OGF in several lines resulted in areduction in cell numbers greater than of either compound alone. In vivothe reduction in tumor volume and weight was synergistic and it appearedthat the OGF reduced the toxicity of paclitaxel resulting in a highersurvival rate.

Carboplatin also resulted in an additive effect reducing squamous cancercell number by 14-27%.

As can be seen the benefits of combination therapy of OGF withchromotherapeutic agents results in a greater reduction in cell numberthan either compound alone, is often synergistic and can also reducetoxicity of the chemotherapeutic agent. This was seen in at least twovery different types of cancer cells in multiple cell lines withdifferent chemotherapeutic agents and both in vitro and in vivo.

It is believed, without being bound by any particular theory, that OGFmay confer protective effects against the cytotoxicity encountered withsome chemotherapeutic agents, such as paclitaxel.

Both OGF and the OGFr have been detected in epithelium of rodent andhuman tongue, skin, gastrointestinal tract, and cornea. It has beenshown that both OGF and the OGFr are present in human tumors whenobtained at the time of surgical resection. Additionally, DNA synthesisof epithelial cells in mammalian tongue, epidermis, cornea and esophagushas been shown to be regulated by OGF, and does so in areceptor-mediated fashion.

OGF has been found to be associated with a reduction in cell number,suggesting that a target of OGF is cell replication. Using humanpancreatic cancer cells in tissue culture, and administering sufficientquantities of OGF to elicit responses that presumably are similar tothose occurring with endogenous OGF, it has been confirmed that OGFrepresses cell accumulation and manifests this activity withintwenty-four hours after OGF exposure. It is believed, without beingbound by any particular theory, that OGF significantly reduces DNAsynthesis and suppresses mitosis, thus modulating cellular generation.

The cell cycle is composed of five phases: the presynthetic or G₁ phase;synthesis of DNA or S phase; post synthetic or G₂ phase (this phasecontains double complement of DNA dividing into two daughter G₁ cells);and mitosis or M phase. Newly divided cells may reenter the cycle or gointo a resting or G₀ phase. OGF has been shown to alter the proportionof cells in phases of the cell cycle so that within about two hoursthere is a marked increase in the number of cells in G₀/G₁ and acompensatory decrease in cells in the S and G₂/M phases. Moreover, OGFappears to increase dramatically the length of the G₀/G₁ phase, thusaccounting for the notable increase in doubling time of the total cellcycle that is observed. It is believed, without being bound by thetheory, that treatment with OGF, either prior to or during radiationtherapy, sensitizes the effect of radiation on tumor cells via theability of OGF to accumulate cancer cells in the G₀/G₁ phase of the cellcycle, where they are most vulnerable to radiation.

Gemcitabine is a pyrimidine analogue that belongs to a general group ofchemotherapy drugs known as antimetabolites that also acts as aradiation-sensitizing agent. Gemcitabine exhibits cell phasespecificity, primarily killing cells undergoing DNA synthesis, i.e., theS-phase, and also blocks the progression of cells through the G₁/S-phaseboundary. Gemcitabine is metabolized intracellularly by nucleosidekinases to the active gemcitabine diphosphate (dFdCDP) and triphosphate(dFdCTP) nucleosides. The cytotoxic effect of gemcitabine is attributedto a combination of two actions of the diphosphate and the triphosphatenucleosides, which leads to inhibition of DNA synthesis. First,gemcitabine diphosphate inhibits ribonucleotide reductase, which isresponsible for catalyzing the reactions that generate thedeoxynucleoside triphosphates for DNA synthesis. Inhibition of thisenzyme by the diphosphate nucleoside causes a reduction in theconcentrations of deoxynucleotide, including dCTP. Second, gemcitabinetriphosphate competes with dCTP for incorporation into DNA. Thereduction in the intracellular concentrations of dCTP (by the action ofthe diphosphate) enhances the incorporation of gemcitabine triphosphateinto DNA (self-potentiation). After the gemcitabine nucleotide isincorporated into DNA, only one additional nucleotide is added to thegrowing DNA strands. After this addition, there is inhibition of furtherDNA synthesis. DNA polymerase epsilon is unable to remove thegemcitabine nucleotide and repair the growing DNA strands (masked chaintermination). In lymphoblastoid cells, gemcitabine inducesinternucleosomal DNA fragmentation, one of the characteristics ofprogrammed cell death.

Paclitaxel, also known as Taxol, is derived from the bark and leaves ofthe Pacific yew (another source is from the needles of a European yew).Paclitaxel is very lipid soluble and must be administered intravenouslysoon after preparation. Paclitaxel is an antimicrotubule agent thatpromotes the assembly of microtubulin dimers and stabilizes microtubulesby preventing depolymerization. This stability results in the inhibitionof the normal dynamic reorganization of the microtubule network that isessential for vital interphase and mitotic cellular functions. Inaddition, paclitaxel induces abnormal arrays or “bundles” ofmicrotubules throughout the cell cycle and multiple asters ofmicrotubules during mitosis. Paclitaxel side effects include transientbradycardia, peripheral neuropathy, nausea, vomiting, diarrhea,neutropenia, thrombocytosis, bronchospasm, urticaria, angioedema,alopecia and myalgias. Premedication with dexamethasone,diphenhydramine, and H2 antagonists are used to reduce hyposensitivityreactions.

Carboplatin and cisplatin belong to the platin family ofchemotherapeutic agents, inorganic platinum complexes that disrupt theDNA helix by forming intra- and interstrand cross-links. Cisplatin inparticular reacts with nucleophils of other tissues, hence its toxiceffect on the kidney, the eight cranial nerve, and the intense emesis.

Both carboplatin and cisplatin are concentrated in the kidney, liver,intestines and testes, but they do not cross the blood brain barrier.They are usually used with other agents in metastatic testicular,ovarian carcinoma, and advanced bladder cancer. Side effects arecommonly encountered with cisplatin administration, and include renaltoxicity, ototoxicity manifested by tinnitus and hearing loss, markednausea and vomiting. Additionally, mild to moderate myelosuppression maydevelop. Carboplatin differs from cisplatin mainly in side effects, asmyelosuppression is the dose-limiting toxicity for carboplatin with verylittle of renal, neurologic, or ototoxicity.

5-FU as a single agent has an activity superior to that of any othersingle agent in the treatment of carcinomas of the colon and rectum. Itis used primarily for slowly growing solid tumors, such as carcinomas ofthe breast and the gastrointestinal tract. The mean response rate isstill low, however, being less than 20%. Inactive as such, fluorouracilmust be converted to the 5′-monophosphate nucleotide where it mayinactivate enzymes essential to synthesize thymidylate, or where it actswithin a complex pathway. 5-FU is incorporated into RNA and inhibits DNAsynthesis. 5-FU is converted into the active 5-fluoro-deoxyuridinemonophosphate (FdUMP) by a variety of different metabolic pathways. Thedrug acts by inhibiting the enzyme thymidylate kinase which results inreduced formation of thymidine and thus of DNA. Fluorouracil, as FdUMP,is also incorporated into RNA, which results in fluoridation of the RNA.

The effect of 5-FU on living cells is limited mainly to those in theproliferative phase. However, while cells in the G₂ and S phases aremost affected there may be effects at any stage of the cell cycle. 5-FUis metabolized primarily in the liver, with only 10% of the drugappearing unchanged in urine. 5-FU can enter cerebrospinal fluid.Resistance to 5-FU develops because the cells lose their ability toconvert 5-FU to its active form. Common side effects are often delayed.Stomatitis that ulcerates is an early sign of toxicity, andmyelosuppression (leukopenia) usually occurs between nine and fourteendays of therapy. Other side effects include alopecia, dermatitis, andatrophy of the skin.

It is believed, without being bound by any particular theory, that thecarcinotherapeutic composition of the present invention, i.e., combiningat least one chemotherapeutic agent with the biotherapeutic agent, OGF,exerts its potent inhibitory effect on cancer cell growth by the abilityof OGF to accumulate cells in the G₀/G₁ phase, where the cells arevulnerable to the cytotoxic effects of a chemotherapeutic agent, thusgreatly enhancing the number of cells killed by the chemotherapeuticagent. A lowered effective dose of the chemotherapeutic agent is needed,therefore, to produce a significantly greater growth inhibition thanwhat would occur without the presence of OGF.

The following non-limiting examples describe in more detail the effectsof administering OGF in combination with gemcitabine, paclitaxel,carboplatin and 5-FU on a human squamous cell carcinoma cell line (SCC-1cell line) and on a human pancreatic cancer cell line (MiaPaCa-2 cellline).

EXAMPLE 1 Combination Therapies on the SCC-1 Cell Line

1. Growth Curves—SCC-1 Cell Lines

The growth of cells as represented by absorbency taken at 450 nm fromthe cell proliferation assay plotted against time was the standardformat for presenting the effects of different drugs on SCC-1 orMiaPaCa-2 cells. Cells were counted using a standard MTT assay. Ingeneral, each data point represents the average absorbency taken from 10wells/treatment; error bars represent the S.E.M.

a. Paclitaxel Treatment

The results illustrated in FIG. 1 and Table I examine the addition ofpaclitaxel (Taxol) and/or OGF to SCC-1 cells. Throughout the 4-daygrowth curve, statistical analysis (ANOVA) revealed that OGF (10⁻⁶ M)alone inhibited growth at 48, 72 and 96 hours decreasing cell numberfrom control levels by 11.9, 6.7, and 12.7%, respectively. Paclitaxel ata concentration of 10⁻⁷ M inhibited cell growth at 24, 48, 72, and 96hours decreasing cell number from controls by 14.2, 34.4, 58, and 70%,respectively. Paclitaxel at a concentration of 10-8 also inhibitedgrowth at 72 and 96 hours with decreases in cell number relative tocontrols of 14.1 and 19.3%, respectively. When paclitaxel 10⁻⁷ wascombined with OGF 10⁻⁶ M, growth inhibition was observed at 24, 48, 72,and 96 hours resulting in a decrease in cell number relative to controlsof 13.5, 52.6, 68.4, and 78.4%, respectively. When paclitaxel 1-8 M wascombined with OGF 10⁻⁶ M, cell growth inhibition was observed at 48, 72,and 96 hours with decreases in cell number relative to controls of 13.6,30.6, and 36.3%, respectively. OGF in combination with paclitaxel 10⁻⁷ Mwas significantly more inhibitory than any drug alone at all timepoints(besides paclitaxel 10⁻⁷ M at 24 hours) with decreases in cell numberranging from 10.1-75.3%. OGF in combination with paclitaxel 10⁻⁸ M wassignificantly more inhibitory than any drug alone at 48, 72, and 96hours with decreases in cell number ranging from 10.4-27.1%.

b. Carboplatin Treatment

The results illustrated in FIG. 2 and Table 2 examine the addition ofcarboplatin and/or OGF to SCC-1 cells. Throughout the 4-day period oftreatment, statistical analysis (ANOVA) revealed that the OGF (10⁻⁶ M)alone inhibited growth at 48, 72, and 96 hours with decreases in cellnumber relative to controls of 8.2, 8.4, and 9.7%, respectively.Carboplatin at a concentration of 10⁻⁶ M inhibited cell growth at 48,72, and 96 hours decreasing cell number relative to controls by 5.3,21.8, and 24.9%, respectively. Carboplatin at a concentration 10⁻⁷ Malso inhibited growth at 72 and 96 hours decreasing cell number relativeto controls by 18.7 and 21%, respectively. When carboplatin 10⁻⁶ M wascombined with OGF 10⁻⁶ M, growth inhibition was observed at 24, 48, 72,and 96 hours resulting in decreases in cell number relative to controlsof 10.3, 17.5, 32.2, and 33.3%, respectively. When carboplatin 10⁻⁷ Mwas combined with OGF 10⁻⁶ M, cell growth inhibition was observed at 48,72, and 96 hours with decreases in cell number relative to controls of14.1, 26.3, and 27.1%, respectively. OGF in combination with carboplatin10⁻⁶ M was significantly more inhibitory than any drug alone at 48, 72,and 96 hours with decreases in cell number ranging from 3.1-23.6%. OGFin combination with carboplatin 10⁻⁷ M was significantly more inhibitorythan OGF alone at 48, 72, and 96 hours, carboplatin 10⁻⁶ M at 48 and 72hours, and carboplatin 10⁻⁷ at 48 and 72 hours with decreases in cellnumber ranging from 10.4-27.1% (Table 2).

c. Body Weights, Life-Span, and Gross Observation

At the beginning of the trial, all mice weighed approximately 22-24grams and mice gained roughly 2 to 4 grams every 5 days. However, by day20 of the experiment, paclitaxel mice began to lose weight, weighing 11%less than controls (p<0.05). Continued weight loss was observed withinthe paclitaxel group until termination day or the death of the mice (seesurvival curve FIG. 5), on day 50 mice weighed 28% less (p<0.001) thancontrols, OGF, and paclitaxel/OGF treated mice (see FIG. 4).

Mice in the paclitaxel group began dying on day 19 (see FIG. 5). By day40, 75% of the paclitaxel treated mice had died and no mouse in anyother treatment group, including the paclitaxel/OGF group had perished.On termination day, only one mouse (8% of the group) was still alive.The average life span of the paclitaxel mice was 34.3±3.1 days and thiswas significantly (p<0.001) different from all other treatment groups.One mouse in the paclitaxel/OGF group died on day 40 but all remainingmice were still alive until termination day.

Due to the premature death of the paclitaxel mice, organs were harvestedand fixed in formalin for histological analysis. Upon analysis, it wasobserved that premature death could have been attributed to distendedabdomens with associated megacolon. The large intestine, cecum, andsmall bowel were all completely impacted with hardened stool. All otherorgan systems appeared normal.

EXAMPLE 2 SCC-1 Tumor Appearance and Growth

All mice that were injected with SCC-1 cells developed tumors. On day 13after tumor cell inoculation, 75% of mice, 66% of paclitaxel treatedmice, and 58% of paclitaxel/OGF treated mice had tumors. When examininglatency to a visible tumor, control mice developed visible, but notmeasurable tumors within 7 days of tumor cell inoculation. Paclitaxeland paclitaxel/OGF mice also developed visible tumors within the same1-week time frame while OGF mice developed tumors within 11 days,exhibiting an approximate 4-day delay in visible tumor development(p<0.05). The latency time for measurable (62.5 mm³) tumors displayed ananalogous pattern to the latency for visible tumors where control,paclitaxel, and paclitaxel/OGF groups had measurable tumors within 2weeks of tumor cell inoculation while the OGF group developed measurabletumors within 17 days, although this difference was not significant fromcontrol values.

Tumor dimensions were recorded every day beginning on the day that thetumors were considered measurable. These were plotted for every 2consecutive days of measurements beginning on the first day that eachmouse had a measurable tumor over the course of 36 days (FIG. 3).

Using data that were platted for every 2 consecutive days ofmeasurements beginning on the first day that each mouse had a measurabletumor over the course of 36 days (FIG. 3), the second timepoint ofmeasurable tumor appearance (4^(th) of measurable tumors), both OHGF andpaclitaxel/OGF mice had significantly (p<0.05) smaller tumors thancontrol mice with reductions of 26% and 29%, respectively. On the 3^(rd)timepoint, all 3 treatment groups had mean tumor volumes that weresignificantly smaller than controls by 29 to 33%. At timepoints 8 to 10,paclitaxel/OGF mice had tumors that were significantly smaller thantumor sizes in groups receiving single treatments. From timepoint 11(see FIG. 3) through the end of the trial, paclitaxel/OGF mice exhibitedtumor volumes that were significantly smaller than both the control andOGF mice, but comparisons to paclitaxel mice revealed no significancedue to the fact that mice in the paclitaxel group began to die aroundthis timepoint. Death of the paclitaxel mice made the statisticalanalysis difficult. In some cases the mice began to exhibit common sideeffects of the chemotherapy and tumor sizes often decreased. Thereforetumor measurements comparing the paclitaxel and paclitaxel/OGF mice wereoften non-significant, both due to the decreased tumor size before deathand lowered N value in the paclitaxel group.

EXAMPLE 3 Combination Therapies on the MiaPaCa-2 Cell Lines

The results illustrated in FIG. 7 examine the addition of 5-FU and/orOGF to MiaPaCa-2 cells. Throughout the 4-day treatment periodstatistical analysis (ANOVA) revealed that the OGF (10⁻⁶ M) alonesignificantly inhibited growth at 24, 48, 72, and 96 hours withdecreases in cell number from controls of 7.1, 7.0, 6.9 and 14.2%,respectively. 5-FU at a concentration of 10-5 M inhibited cell growth at48, 72, and 96 hours decreasing cell number by 26.0, 30.1, and 36.4%,respectively relative to controls. 5-FU at a concentration of 10⁻⁶ Malso inhibited growth at 48, 72 and 96 hours decreasing cell numberrelative to controls by 12.7, 10.8 and 15.2%, respectively. When 5-FU(10⁻⁵ M) was combined with OGF (10⁻⁶ M), growth inhibition was observedat 24, 48, 72, and 96 hours resulting in decreases in cell number fromsterile water treated controls of 21.5, 35.7, 39.7, and 47.4%,respectively. When 5-FU (10⁻⁶ M) was combined with OGF (10⁻⁶ M), cellgrowth inhibition was observed at 24, 48, 72 and 96 hours with decreasesin cell numbers from control of 13.2, 22.2, 23.6 and 30.3%,respectively. OGF in combination with 5-FU at 10⁻⁵ M was significantlymore inhibitory than any drug alone at 24, 48, 72 and 96 hours, withdecreases in cell numbers ranging from 15.5 to 38.7%.

EXAMPLE 4 Gemcitabine and OGF Cell Cycle Phase Analysis

To investigate the exact cell cycle phase where gemcitabine and/or OGFexerted their effects, flow cytometry was performed. OGF showed nosignificant increases into the G₀/G₁ phase of the cell cycle althoughslight increases in the percentages of cells in this phase were observedat 2, 4, 6, 8, 12, 20, and 24 hours after OGF exposure. Gemcitabine isknown to alter the G₁/S phase and this recruitment can be observed asearly as 6 hours after treatment with either gemcitabine orgemcitabine/OGF. Decreased percentages of cells in the G₂/M phase of thecell cycle were observed with cells treated with gemcitabine/OGF ascompared to cells treated with just gemcitabine, indicating that morecells were stalled in the G₁S with the combined therapy. At 48 and 120hours of treatment with gemcitabine, G₁ recruitment remained strong with73.50% and 60.75% of cells respectively still in G₁ with thegemcitabine/OGF treatment at 48 and 120 hours, 74.03% and 60.15% ofcells were arrested in the G₁ phase of the cell cycle.

EXAMPLE 5 In Vivo OGF/Gemcitabine Treatment in Nude Mouse Model

To examine the effectiveness of the combined OGF/gemcitabine treatmentsin vivo, an athymic nude mouse model was used. Treatments of OGF (10mg/kg daily), gemcitabine (120 mg/kg every 3 days), and gemcitabine (120mg/kg every 3 days)/OGF (10 mg/kg daily) were used to treat the miceinoculated with 1×10⁶ MiaPaCa-2 cells.

a. MiaPaCa-2 Tumor Appearance and Growth

All mice that were injected with MiaPaCa-2 cells developed tumors. Onday 16 after tumor cell inoculation, all mice in the control salinetreatment group as well as the OGF group had a tumor, while 75% ofgemcitabine treated mice, and 0% of gemcitabine/OGF (p<0.0001) treatedmice, had tumors (See FIG. 8). When examining latency to a visibletumor, control mice developed visible, but not measurable, tumors within10 days of tumor cell inoculation. OGF mice and gemcitabine mice alsodeveloped tumors within the same 10-day time frame while gemcitabine/OGFmice developed tumors within 16 days, exhibiting an approximate 6-daydelay in visible tumor development (p<0.05). The latency time formeasurable (62.5 mm³) tumors displayed an analogous pattern to thelatency for visible tumors. Control, OGF, and gemcitabine groups hadmeasurable tumors within 2 weeks of tumor cell inoculation while thegemcitabine/OGF group developed measurable tumors within 20 days(p<0.05). Tumor dimensions were recorded every day beginning on the daythat the tumors were considered measurable and data were plotted forevery 2 consecutive days of measurements beginning on the first day thateach mouse had a measurable tumor over the course of 31 days.

Using data that were plotted for every 2 consecutive days ofmeasurements beginning on the first day that each mouse had a measurabletumor over the course of 36 days (FIG. 8) from the 6^(th) timepoint ofmeasurable tumor appearance (12^(th) day of measurable tumor incidence),OGF mice had significantly (p<0.01 timepoints 7, 9, and 16, p<0.001timepoint 8, and p<0.05 all remaining) smaller tumors than control micewith reductions of 29.9-40.7%, respectively. Starting with the 4^(th)timepoint, all 3 treatment groups had tumors that were significantlysmaller than control tumors. At every timepoint besides 3, 9, 10, and11, gemcitabine/OGF mice had tumors that were significantly smaller thantumor sizes in the OGF group. At timepoints 1, 4, 14, 15, and 16,gemcitabine/OGF mice had significantly smaller tumors than thegemcitabine mice alone. Tumor volumes of mice receiving gemcitabine onlysignificantly differed from the tumor volumes of mice receiving OGF attimepoints 4, 5, and 6. Although ANOVA did not reveal many significancesbetween gemcitabine versus gemcitabine/OGF other than mentioned above,volumes of gemcitabine/OGF tumors were smaller by 29.8-56.9% at pointsthat were not deemed significant by ANOVA.

EXAMPLE 6 Gemcitabine Growth Curve-Cell Counting

Further investigation of the effects of gemcitabine and/or OGF on thegrowth of MiaPaCa-2 cells was explored by performing actual cell counts.FIG. 9 illustrates that OGF (10⁻⁶ M) alone inhibited growth at 48, 72,and 96 hours with decreases in cell number from controls of 15.5, 17.6,and 16.7%, respectively. Gemcitabine at a concentration of 10⁻⁷ Minhibited cell growth at 24, 48, 72, and 96 hours decreasing cell numberrelative to controls by 30.1, 46.4, 47.7, and 64.2%, respectively.Gemcitabine at a concentration of 10⁻⁸ M also inhibited growth at 48,72, and 96 hours decreasing cell number relative to controls by 21.7,21.2, and 32.4%, respectively. When gemcitabine (10⁻⁸ M) was combinedwith OGF (10⁻⁶ M), growth inhibition was observed at 48, 72, and 96hours resulting in decreases in cell number of 26.3, 49.2, and 45.9%,respectively. Gemcitabine (10⁻⁸M) when combined with OGF (10⁻⁶ M) wassignificantly more inhibitory than OGF alone at 72 and 96 hours andgemcitabine (10⁻⁸ M) alone at 72 and 96 hours (See Table 5).

Table 1 shows significance values obtained from a one-way ANOVA forpaclitaxel and/or OGF versus controls (A), OGF (B), paclitaxel 10⁻⁷ M(C), and paclitaxel 10⁻⁸ M (D) over a 96-hour trial.

Table 2 shows significance values obtained from a one-way ANOVA forcarboplatin (Carb) and/or OGF versus controls (A), OGF (B), carboplatin10⁻⁶ M (C), and carboplatin 10⁻⁷ M (D) over a 96-hour trial.

Table 3 shows significance values obtained from a one-way ANOVA forgemcitabine and/or OGF versus controls (A), OGF 10⁻⁶ M (B), gemcitabine10⁻⁷ M (C), and gemcitabine 10⁻⁸ M (D) over a 96-hour trial.

Table 4 shows significance values obtained from a one-way ANOVA for 5-FUand/or OGF versus controls (A), OGF (B), 5-FU 10⁻⁵ M (C), or 5-FU 10⁻⁶ M(D) over a 96-hour trial.

Table 5 shows significance values obtained from a one-way ANOVA forgemcitabine and/or OGF versus controls (A), OGF (B), gemcitabine 10⁻⁷ M(C), and gemcitabine 10⁻⁸ M (D) over a 96-hour trial. TABLE 1Significance values obtained from a one-way ANOVA for paclitaxel (Taxol[Tax]) and/or OGF versus controls (A), OGF (B), paclitaxel 10⁻⁷ M (C),and paclitaxel 10⁻⁸ M (D) over a 96-hour trial. Hours [OGF] [Tax 10⁻⁷M][Tax 10⁻⁸M] [Tax 10⁻⁷M/OGF] [Tax 10⁻⁸M/OGF] A Significance from ControlValues 24 *** *** 48 ** *** *** *** 72 ** *** *** *** *** 96 *** *** ****** *** B Significance from OGF Values 24 ++ ++ 48 +++ +++ ++ 72 +++ +++++ +++ 96 +++ +++ +++ C Significance from Taxol 10⁻⁷ M Values 24{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a) 48{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a){circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a) 72{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a){circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a) 96{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a){circumflex over ( )}{circumflex over ( )} {circumflex over( )}{circumflex over ( )}{circumflex over ( )}^(a) D Significance fromTaxol 10⁻⁸ M Values 24 ### ### 48 ### ### ### 72 ### ### ### 96 ### ######Significantly different from controls at p < 0.001 (***), p < 0.01 (**),and p < 0.05 (*).Significantly different from OGF at p < 0.001 (+++), p < 0.01 (++), andp < 0.05 (+).Significantly different from Taxol 10⁻⁷ M at p < 0.001 ({circumflex over( )}{circumflex over ( )}{circumflex over ( )}), p < 0.01 ({circumflexover ( )}{circumflex over ( )}), p < 0.05 ({circumflex over ( )}).Significantly different from Taxol 10⁻⁸ M at p < 0.001 (###), p < 0.01(##), p < 0.05 (#).^(a)Indicates that Taxol 10⁻⁸ M and Taxol 10⁻⁸ M/OGF was significantly(p < 0.001) less inhibitory than Taxol 10⁻⁷ M at these timepoints.

TABLE 2 Significance values obtained from a one-way ANOVA forcarboplatin (Carb) and/or OGF versus controls (A), OGF (B), carboplatin10⁻⁶ M (C), and carboplatin 10⁻⁷ M (D) over a 96-hour trial. Hours [OGF][Carb 10⁻⁶ M] [Carb 10⁻⁷ M] [Carb 10⁻⁶ M/OGF] [Carb 10⁻⁷ M/OGF] ASignificance from Control Values 24 ** 48 *** ** *** *** 72 *** *** ****** *** 96 *** *** *** *** *** B Significance from OGF Values 24 48+++^(a) ++ +++ 72 +++ +++ +++ +++ 96 +++ +++ +++ +++ C Significance fromCarboplatin 10⁻⁶ M Values 24 48 {circumflex over ( )}^(b) {circumflexover ( )}{circumflex over ( )}{circumflex over ( )} {circumflex over( )}{circumflex over ( )}{circumflex over ( )} 72 {circumflex over( )}{circumflex over ( )}^(b) {circumflex over ( )}{circumflex over( )}{circumflex over ( )} {circumflex over ( )} 96 {circumflex over( )}{circumflex over ( )} D Significance from Carboplatin 10⁻⁷ M Values24 48 # ### ### 72 ## ### ### 96 ###Significantly different from controls at p < 0.001 (***), p < 0.01 (**),and p < 0.05 (*).Significantly different from OGF at p < 0.001 (+++), p < 0.01 (++), andp < 0.05 (+).Significantly different from carboplatin 10⁻⁶ M at p < 0.001({circumflex over ( )}{circumflex over ( )}{circumflex over ( )}), p <0.01 ({circumflex over ( )}{circumflex over ( )}), p < 0.05 ({circumflexover ( )}).Significantly different from carboplatin 10⁻⁷ M at p < 0.001 (###), p <0.01 (##), p < 0.05 (#).^(a)Indicates that OGF was significantly (p < 0.001) more inhibitorythan carboplatin 10⁻⁷ M at this timepoint.^(b)Indicates that carboplatin 10⁻⁶ M was significantly more inhibitorythan carboplatin 10⁻⁷ M at these timepoints.

TABLE 3 Significance values obtained from a one-way ANOVA forgemcitabine and/or OGF versus controls (A), OGF 10⁻⁶ M (B), gemcitabine10⁻⁷ M (C), and gemcitabine 10⁻⁸ M (D) over a 96-hour trial. Hours [OGF][Gem 10⁻⁷ M] [Gem 10⁻⁸ M] [Gem 10⁻⁷ M/OGF] [Gem 10⁻⁸ M/OGF] ASignificance from Control Values 24 ** *** * 48 *** *** *** 72 *** ****** *** *** 96 *** *** *** *** *** B Significance from OGF Values 24 +++48 +++ ++ +++ 72 +++ +++ +++ 96 +++ ++ +++ +++ C Significance fromGemcitabine 10⁻⁷ M Values 24 {circumflex over ( )}{circumflex over( )}{circumflex over ( )} 48 {circumflex over ( )}{circumflex over( )}{circumflex over ( )}^(a) {circumflex over ( )}{circumflex over( )}{circumflex over ( )} 72 {circumflex over ( )}{circumflex over( )}{circumflex over ( )}^(a) {circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a) 96{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a){circumflex over ( )}{circumflex over ( )} {circumflex over ( )}^(a) DSignificance from Gemcitabine 10⁻⁸ M Values 24 ### 48 ###^(b) ### ### 72###^(b) ### ### 96 ###^(b) ### ###Significantly different from controls at p < 0.001 (***), p < 0.01 (**),and p < 0.05 (*).Significantly different from OGF at p < 0.001 (+++), p < 0.01 (++), andp < 0.05 (+).Significantly different from Gemzar 10⁻⁷M at p < 0.001 ({circumflex over( )}{circumflex over ( )}{circumflex over ( )}), p < 0.01 ({circumflexover ( )}{circumflex over ( )}), p < 0.05 ({circumflex over ( )}).Significantly different from Gemzar 10⁻⁸M at p < 0.001 (###), p < 0.01(##), p < 0.05 (#).^(a)Indicates that Gemzar 10⁻⁸M and Gemzar 10⁻⁸M/OGF was significantly(p < 0.001 {circumflex over ( )}{circumflex over ( )}{circumflex over( )}, p < 0.05{circumflex over ( )}) less inhibitory than Gemzar 10⁻⁷Mat these timepoints.^(b)Indicates that Gemzar 10⁻⁸M was significantly (p < 0.001) lessinhibitory than Gemzar 10⁻⁷M at these timepoints.

TABLE 4 Significance values obtained from a one-way ANOVA for 5-FUand/or OGF versus controls (A), OGF (B), 5-FU 10⁻⁵ M (C), or 5-FU 10⁻⁶ M(D) over a 96-hour trial. Hours [OGF] [5-FU 10⁻⁵ M] [5-FU 10⁻⁶ M] [5-FU10⁻⁵ M/OGF] [5-FU 10⁻⁶ M/OGF] A Significance from Control Values 24 ****** *** 48 * *** *** *** *** 72 ** *** *** *** *** 96 *** *** *** ****** B Significance from OGF Values 24 ++^(a) ++^(a) +++ +++ 48 +++ ++++++ 72 +++ +++ +++ 96 +++ +++ +++ C Significance from 5-FU 10⁻⁵ M Values24 {circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(b) 48{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(b){circumflex over ( )}{circumflex over ( )} 72 {circumflex over( )}{circumflex over ( )}{circumflex over ( )}^(b) {circumflex over( )}{circumflex over ( )}{circumflex over ( )} {circumflex over ( )}^(b)96 {circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(b){circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(b) D Significance from 5-FU 10⁻⁶ M Values 24 ###### 48 ###^(b) ### ## 72 ###^(b) ### ### 96 ###^(b) ### ###Significantly different from controls at p < 0.001 (***), p < 0.01 (**),and p < 0.05 (*).Significantly different from OGF at p < 0.001 (+++), p < 0.01 (++), andp < 0.05 (+).Significantly different from 5-FU 10⁻⁵ M at p < 0.001 ({circumflex over( )}{circumflex over ( )}{circumflex over ( )}), p < 0.01 ({circumflexover ( )}{circumflex over ( )}), p < 0.05 ({circumflex over ( )}).Significantly different from 5-FU 10⁻⁶ M at p < 0.001 (###), p < 0.01(##), p < 0.05 (#).^(a)Indicates that OGF was significantly (p < 0.001) more inhibitorythan 5-FU 10⁻⁵ M and 5-FU 10⁻⁶ M at these timepoints.^(b)Indicates that 5-FU 10⁻⁵ M was significantly more inhibitory than5-FU 10⁻⁶ M and 5-FU 10⁻⁶ M/OGF 10⁻⁶ M at these timepoints.

TABLE 5 Significance values obtained from a one-way ANOVA forgemcitabine and/or OGF versus controls (A), OGF 10⁻⁶ M (B), gemcitabine10⁻⁷ M (C), and gemcitabine 10⁻⁸ M (D) over a 96-hour trial. Hours [OGF][Gem 10⁻⁷ M] [Gem 10⁻⁸ M] [Gem 10⁻⁷ M/OGF] [Gem 10⁻⁸ M/OGF] ASignificance from Control Values 24 ** *** * 48 *** *** *** 72 *** ****** *** *** 96 *** *** *** *** *** B Significance from OGF Values 24 +++48 +++ ++ +++ 72 +++ +++ +++ 96 +++ ++ +++ +++ C Significance fromGemcitabine 10⁻⁷ M Values 24 {circumflex over ( )}{circumflex over( )}{circumflex over ( )} 48 {circumflex over ( )}{circumflex over( )}{circumflex over ( )}^(a) {circumflex over ( )}{circumflex over( )}{circumflex over ( )} 72 {circumflex over ( )}{circumflex over( )}{circumflex over ( )}^(a) {circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a) 96{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}^(a){circumflex over ( )}{circumflex over ( )} {circumflex over ( )}^(a) DSignificance from Gemcitabine 10⁻⁸ M Values 24 ### 48 ###^(b) ### ### 72###^(b) ### ### 96 ###^(b) ### ###Significantly different from controls at p < 0.001 (***), p < 0.01 (**),and p < 0.05 (*).Significantly different from OGF at p < 0.001 (+++), p < 0.01 (++), andp < 0.05 (+).Significantly different from Gemzar 10⁻⁷M at p < 0.001 ({circumflex over( )}{circumflex over ( )}{circumflex over ( )}), p < 0.01 ({circumflexover ( )}{circumflex over ( )}), p < 0.05 ({circumflex over ( )}).Significantly different from Gemzar 10⁻⁸M at p < 0.001 (###), p < 0.01(##), p < 0.05 (#).^(a)Indicates that Gemzar 10⁻⁸ and Gemzar 10⁻⁸M/OGF was significantly (p< 0.001) less inhibitory than Gemzar 10⁻⁷M at these timepoints.^(b)Indicates that Gemzar 10⁻⁸M was significantly (p < 0.001) lessinhibitory than Gemzar 10⁻⁷M at these timepoints.

EXAMPLE 7

Given the promising nature of OGF (biotherapy), and of gemcitabine(chemotherapy), as antitumor agents in pancreatic cancer, and the lackof preclinical data regarding the simultaneous use of OGF andgemcitabine, the present study was designed to explore the therapeuticpotential of a combination of these modalities. Using a tissue culturemodel of human pancreatic adenocarcinoma, the effect of concomitantexposure to both OGF and gemcitabine were characterized on growth (e.g.,reversibility, receptor mediation, specificity). The relationship ofanother chemotherapy treatment (i.e., 5-FU) and OGF in regard topancreatic cancer, as well as the ubiquity of combined therapy on otherpancreatic cancer cell lines, were evaluated. Finally, the presentreport addresses the question of whether a combination of OGF andgemcitabine influences growth of human pancreatic cancer in vivo, anddoes so beyond the efficacy of each compound. The effects of OGF and/orgemcitabine on tumor incidence, appearance, and size, as well asmetastasis, were examined in a xenograft model of pancreatic cancer.

Material and Methods

Cell Lines

MIA PaCa-2 and PANC-1 human pancreatic adenocarcinoma cell lines werepurchased from the American Type Culture Collection (Manasass, Va.). MIAPaCa-2 cells were derived from an undifferentiated epithelial carcinomaoccurring in the body and tail of the pancreas in a 65-year-old man[36]. The PANC-1 cells were derived from an undifferentiated carcinomafrom the head of the pancreas in a 56-yr old man [18]. MIA PaCa-2 andPANC-1 cells were grown in Dulbecco's MEM (modified) media; media wassupplemented with 10% fetal calf serum, 1.2% sodium bicarbonate, andantibiotics (5,000 Units/ml penicillin, 5 mg/ml streptomycin, 10 mg/mlneomycin), and the cells were maintained in a humidified atmosphere of7% CO₂/93% air at 37° C.

Growth Assays

MIA PaCa-2 cells were seeded at equivalent amounts into either 75 cm²flasks, 6-well plates, or 96-well plates (Falcon) and counted 24 hrlater to determine plating efficiency. Growth assays for PANC-1 cellswere conducted in 6-well plates (Falcon). Compounds or sterile waterwere added beginning 24 hr after seeding (=0 hr), and both media andcompounds were replaced daily. All drugs were prepared in sterile waterand dilutions represent final concentrations of the compounds.

Cell number was recorded either by using a mitogenic bioassay, the MTSassay (Cell Titer 96 One Solution, Promega, Madison, Wis.), andmeasuring absorbency after 4 hr on a Biorad (Model 3550) plate reader at490 nm, or by counting cells. For manual counts, cells were harvestedwith a solution of 0.25% trypsin/0.53 mM EDTA, centrifuged, and countedwith a hemacytometer. Cell viability was determined by trypan bluestaining. At least two aliquots per flask or 4-10 wells/treatment werecounted at each time.

Animals and Tumor Cell Implantation

Male 4 week old BALB/c-nu/nu nude mice purchased from HarlanLaboratories (Indianapolis, EN) were housed in pathogen-free isolatorsin the Department of Comparative Medicine at the Penn State UniversityCollege of Medicine. All procedures were approved by the IACUC committeeof the Penn State University College of Medicine and conformed to theguidelines established by NIH. Mice were allowed 48 hr to acclimateprior to beginning experimentation.

MIA PaCa-2 cells (10⁶ cells/mouse) were inoculated into nude mice bysubcutaneous injection into the right scapular region; mice were notanesthetized for this procedure.

Drug Treatment

Four groups of mice (n=12) were randomly assigned to receiveintraperitoneal injections of 10 mg/kg OGF daily, 120 mg/kg gemcitabineevery 3 days; 10 mg/kg OGF daily and 120 mg/kg gemcitabine every 3rdday, or 0.1 ml of sterile saline daily [29, 38]. All drugs weredissolved in saline and prepared weekly. Injections were given within 1hr of tumor cell inoculation.

Tumor Growth and Metastases

Mice were weighed weekly throughout the experiment, and observed dailyfor the presence of tumors. The latency for a visible tumor to appear,and the time until tumors were measurable (i.e., 62.5 mm³) wererecorded. Tumors were measured using calipers every day after tumorappearance. Tumor volume was calculated using the formula w²×1×π/6,where the length is the longest dimension, and width is the dimensionperpendicular to length [31].

Termination Day Measurements

According to IACUC guidelines, mice were terminated when tumors becameulcerated, or tumors grew to 2 cm in diameter. Forty-five days followingtumor cell inoculation, all mice were euthanized by an overdose ofsodium pentobarbital (100 mg/kg) and killed by cervical dislocation;mice (with tumors) were weighed. Tumors and spleens were removed andweighed, and the lymph nodes, liver, and spleen examined for metastases.

Plasma Levels of [Met⁵]-enkephalin (OGF)

At the time of termination, trunk blood was collected from some mice ineach group. Plasma was separated and OGF levels were measured bystandard radioimmunoassay procedures using a [Met⁵]-enkephalin kit fromPeninsula Laboratories (Belmont, Calif.).

Chemicals

The following compounds were obtained from Sigma Chemicals (St. Louis,Mo.): [Met⁵]-enkephalin (OGF), [D-Pen^(2,5)]-enkephalin (DPDPE),[D-Ala²,MePhe⁴,Glyol⁵]-enkephalin (DAMGO), β-endorphin, naltrexone(NTX), naloxone, dynorphin A1-8, [D-Ala-D-Leu-enkephalin] (DADLE),morphine, endomorphin-1, and endomorphin-2.

Data Analysis

Cell numbers and/or absorbencies were analyzed using analysis ofvariance (ANOVA) (one- or two-factor where appropriate) with subsequentcomparisons made using Newman-Keuls tests. Incidence of tumors wasanalyzed by chi-square tests. Latency for tumor appearance and tumorvolume were analyzed using either two-tailed t-tests or ANOVA withsubsequent comparisons made using Newman-Keuls tests. Termination data(i.e., body weight, tumor weight, spleen weight) and OGF plasma levelswere compared by ANOVA.

Results

Growth Assays with OGF and/or Gemcitabine

Growth curves for MIA PaCa-2 cell cultures treated with 10⁻⁶ M OGF (adosage known to inhibit proliferation of MIA PaCa-2 cells, 44), 10⁻⁸ Mgemcitabine (a dosage selected because preliminary experiments revealedno logarithmic growth with a dosage of 10⁻⁷ M), 10⁻⁸ M gemcitabine and10⁻⁶ M OGF, or sterile water (Controls) are presented in FIG. 10. OGFalone inhibited growth at 48, 72, and 96 hr relative to controls, withdecreases in cell number of 16%, 18%, and 17%, respectively, noted.Gemcitabine alone decreased cell number relative to controls at 48, 72,and 96 hr by 22%, 21%, and 32%, respectively. Cells treated with acombination of OGF and gemcitabine were decreased in number relative tocontrols by 26%, 49%, and 46% at 48, 72, and 96 hr, respectively. At 72hr, cell number in cultures receiving the combined therapy ofgemcitabine and OGF was reduced (p<0.001) from cells exposed only to OGFor gemcitabine by 38% and 36%, respectively. Moreover, at 96 hr, thecombined therapy of gemcitabine and OGF reduced (p<0.001) MIA PaCa-2cell number by 35% and 20% from cultures receiving only OGF orgemcitabine, respectively.

Growth Assays with 5-Fluorouracil

To examine whether OGF could enhance the inhibitory effects of otherchemotherapies commonly used to treat pancreatic cancer, MIA PaCa-2 cellcultures were exposed to 5-fluorouracil (5-FU) at a concentration of10⁻⁶ M for 4 days (FIG. 11). MIA PaCa-2 cell number in the 5-FU groupwas reduced 11% to 15% from control levels at 48, 72, and 96 hr.Combination therapy of 5-FU (10⁻⁶ M) and OGF (10⁻⁶ M) reduced cellnumber from control values at 24, 48, 72, and 96 hr by 13% to 30%. Atall time points examined, the combined therapy of 5-FU and OGF reducedMIA PaCa-2 cell number by 6% to 19% from cultures receiving only OGF,and 10% to 17% from cultures receiving only 5-FU.

Receptor Mediated Effects of OGF and/or Gemcitabine

To inquire whether OGF activity was mediated by the OGF receptor, ashort-acting opioid antagonist, naloxone, was added at a dosage of 10⁻⁶Minto cultures receiving 10⁶M OGF and/or gemcitabine (10⁻⁸ M). MIA PaCa-2cells grown in 96-well plates were treated with 10⁻⁶ M OGF, 10⁻⁶ Mnaloxone, 10⁻⁸ M gemcitabine, or combinations at the sameconcentrations—OGF/naloxone, gemcitabine/naloxone, gemcitabine/OGF, andgemcitabine/OGF/naloxone; control cultures received sterile water.Individual plates were read at 96 hr after drug addition. Relative tocontrol levels, addition of OGF, gemcitabine, gemcitabine/OGF, andgemcitabine/OGF/naloxone inhibited cell growth from 13% to 36% (FIG.12). Addition of naloxone completely blocked the growth inhibitoryeffects of OGF alone, but had no effect on the growth inhibitory actionof gemcitabine alone. Moreover, naloxone partially neutralized theenhanced inhibitory effect of the combination of gemcitabine and OGF;cell number of the gemcitabine/OGF/naloxone group was comparable tocells exposed to gemcitabine, but were significantly reduced fromcontrol levels. Naloxone alone had no effect on the growth of MIA PaCa-2cells.

Reversibility of the Inhibitory Growth Effects of OGF and/or Gemcitabine

To establish whether the effect of OGF and/or gemcitabine on cell numbercould be reversed by withdrawing cells from drug exposure, cultures ofMIA PaCa-2 cells were exposed for 48 hr to 10⁻⁶ M OGF and/or 10⁻⁸ Mgemcitabine. At 48 hr after drug exposure, half of the plates had mediaremoved and fresh media added with no addition of OGF or gemcitabine(i.e., OGF-reversal; gemcitabine-reversal; gemcitabine/OGF-reversalgroups); some cultures continued to receive new media and drugs. At 96hr (i.e., 48 hr after reversal), the OGF, gemcitabine,gemcitabine-reversal, gemcitabine/OGF, and the gemcitabine/OGF-reversalgroups differed from controls by 21% to 46% (FIGS. 13A, B). TheOGF-reversal group had 16% more cells than in the OGF group continuingwith OGF exposure. However the gemcitabine-reversal group did not differfrom cell cultures continuing to be treated with gemcitabine. Cellcultures exposed to the combination of OGF and gemcitabine had 7% fewercells than cultures in the gemcitabine/OGF-reversal group.

Specificity of Opioid Peptide(s) Related to Pancreatic Cancer CellGrowth

To determine whether other opioid peptide(s) is(are) related to growth,MIA PaCa-2 cultures (1,000 cells/well) were treated daily with 10⁻⁶ Mconcentrations of a variety of natural and synthetic opioid ligands. Insome cases, these ligands were specific for other opioid receptors(e.g., μ, δ, or κ receptors). Drugs included OGF, DAMGO, morphine,DPDPE, DADLE, dynorphin A1-8, endomorphin-1, endomorphin-2, andβ-endorphin. Cell number was measured on a plate reader after 96 hr oftreatment (both drug and media were changed daily). OGF inhibited cellnumber by 16% relative to controls; none of the other drugs utilized hadany inhibitory or stimulatory effect on growth (FIG. 14).

Ubiquity of Growth Inhibition by OGF

To determine whether the growth inhibition observed with MIA PaCa-2cells following exposure to the combination of gemcitabine and OGF wasnot a cell-line specific action, another human pancreatic cancer cellline, PANC-1, was tested. After 72 hr, exposure of PANC-1 cells toeither OGF (10⁻⁶ M), gemcitabine (10⁻⁸ M), OGF (10⁻⁶ M) and gemcitabine(10⁻⁸ M) revealed 31%, 31%, and 54%, respectively, fewer cells than incontrol cultures (FIG. 15). These differences in cell growth withexposure to OGF and/or gemcitabine differed significantly (p<0.001) fromcontrol levels, and the combination of OGF and gemcitabine differed fromthe OGF alone and the gemcitabine alone cultures at p<0.01.

MIA PaCa-2 Tumor Appearance and Growth

To investigate the effects of OGF and/or gemcitabine on pancreatic tumorgrowth in vivo, nude mice were injected with MIA PaCa-2 cells andtreated with drugs. On day 10, when 80% of the mice in thesaline-injected control group had measurable tumors, and 60% of the OGFand 75% of the gemcitabine-treated animals had tumors, no mouse in thegemcitabine/OGF group had a measurable tumor; the group receivingcombination therapy of gemcitabine and OGF differed significantly fromall other groups at p<0.001 (Table 6). On day 16, no differences in theincidence of measurable tumors could be detected between groups, and allanimals had a tumor by day 17. The latency time for the appearance of avisible tumor in mice of the gemcitabine/OGF group was delayed byapproximately 5 to 6 days from animals in the control, OGF, andgemcitabine groups; this delay for the gemcitabine/OGF group differedsignificantly from that of all other groups at p<0.05. The mean latencytine for measurable tumor appearance in mice of the gemcitabine/OGFgroup was delayed (p<0.05) by approximately 6 days from animals in thecontrol, OGF, and gemcitabine groups.

Changes in tumor volume over the 45 days of the experiment were analyzed(FIG. 16). The OGF, gemcitabine, and gemcitabine/OGF groups all differed(at least p<0.05) from controls in tumor volume beginning on day 14.Tumor volumes of mice receiving combined therapy (i.e., gemcitabine/OGF)differed (p<0.05) from mice treated with only OGF beginning on day 10,and from gemcitabine alone beginning on day 35. Differences in tumorvolumes between groups persisted through the remainder of theexperimental period. Rates of growth over the 45-day period of time wereanalyzed and presented in FIG. 16B. The results demonstrated that thegrowth rates of tumors for all 3 treatment groups were markedly reduced(p<0.001) from control levels. Moreover, the rate of growth of tumors inmice treated with a combination of gemcitabine and OGF weresignificantly decreased (p<0.001) from both the OGF alone and thegemcitabine alone groups.

At the time of termination (i.e., day 45), body weights of all groups ofmice did not differ by statistical evaluation (Table 7). Moreover,autopsy of the animals in each group did not reveal any metastases.However, the weight of the spleen on day 45 for mice in the gemcitabinealone and the gemcitabine/OGF groups were decreased approximately 40%from control values; no changes in spleen weight of the OGF group incomparison to control levels were noted (Table 7). The weights of tumorson the termination day for the OGF alone, gemcitabine alone, andgemcitabine/OGF groups were decreased 36%, 56%, and 85%, respectively,from control levels (Table 7). Tumor volumes on day 45 for the OGFalone, gemcitabine alone, and gemcitabine/OGF groups were decreased 46%,56%, and 83%, respectively, from control values (Table 7).

Plasma Levels of OGF

OGF levels in the plasma of nude mice bearing MIA PaCa-2 tumors rangedfrom 129 to 289 pg/ml. No differences were noted between control miceand those treated with OGF alone, gemcitabine alone, or gemcitabine/OGF.

Discussion

The results in this study demonstrate that the combination of OGF andgemcitabine has a potent inhibitory effect on growth in vitro of humanpancreatic cancer. The antigrowth action of the combination of OGF andgemcitabine was always greater than the individual drugs. In a number ofinstances the effect of the combination of drugs exceeded that of thesum of the individual drugs, suggesting that the action of a combinationof OGF and gemcitabine was supra-additive. The repressive effects ongrowth in vitro of pancreatic cancer cells observed with OGF and withgemcitabine individually were consonant with previous results [e.g., 8,35, 44]. The action of OGF on cell growth was mediated by analoxone-sensitive receptor. This naloxone-sensitive receptor ispresumed to be OGFr, because synthetic and natural opioids selective forclassical opioid receptors such as μ, δ, and κ did not influence growthof pancreatic cancer cells in the present report and earlier [44]. OGFalso was discovered to have a reversible action on the replication ofMIA PaCa-2 cells, supporting the result from earlier studies showingthat treatment with this compound does not lead to cytotoxicity or celldeath [39, 44]. On the other hand, the effects of gemcitabine on MIAPaCa-2 cells were neither blocked by naloxone nor could they bereversed, indicating that the characteristics of this drug's effects onMIA PaCa-2 cells is markedly different from that of OGF. Thus, this isthe first report of the efficacy of using a combination of thebiotherapeutic agent, OGF, and the chemotherapeutic agent, gemcitabine,to retard the growth of human pancreatic cancer.

Although this report concentrated on the effects of OGF and gemcitabineon MIA PaCa-2 cells, it is known that OGF, and gemcitabine, influencethe growth of a variety of human pancreatic cancer cell lines [8, 35,44]. The present investigation demonstrates that not only does OGF andgemcitabine in combination rather than individually have a more markedeffect on MIA PaCa-2 cell growth, but a similar pattern can be foundwith another human pancreatic cancer cell line, PANC-1. Thus, it isreasonable to conclude that the effects of combination therapy with OGFand gemcitabine observed herein also extend to other human pancreaticcancer cell lines.

To address the question of whether OGF could be combined withchemotherapeutic agents other than gemcitabine, a preliminary study wasconducted with the combination of OGF and 5-FU. This allowed a contrastbetween an antimetabolite (5-FU) and a cytosine analogue [32]. Themechanism of 5-FU, a pyrimidine analogue, is to inhibit thymidylatesynthase (an enzyme involved in de novo synthesis of pyrimidines) by theactive metabolite 5-fluoro-deoxyuridine-monophosphate. In addition, theactive triphosphate metabolites, 5-fluoro-deoxyuridine-triphosphate and5-fluoro-uridine-triphosphate, disrupt nucleic acid functions [6]. Thepresent results are the first to show that the effects of a combinationof 5-FU and OGF has potent inhibitory properties with respect to humanpancreatic cancer. As in the case of gemcitabine and OGF, the effect ofa combination of 5-FU and OGF on pancreatic cancer cells was markedlygreater than that of each drug and was often additive in nature.Presumably, these results would indicate that OGF could be used incombination with a variety of chemotherapeutic agents.

The results of this study show that the antigrowth properties of OGF andgemcitabine are enhanced beyond the inhibitory effects of each drugalone. These data were most evident for tumor incidence, latency to avisible or measurable tumor, tumor weight, and tumor volume. Thus, theresults of in vivo studies are consonant with observations conducted invitro. Even though the tumor transplanatation investigation focused onone human pancreatic cancer cell line, it is known that OGF orgemcitabine influences the growth of a variety of human pancreaticcancer cell lines in vivo [3, 28, 35, 38]. Therefore, the effects ofcombination therapy with OGF and gemcitabine shown in this study willextend to other human pancreatic cancer cell lines in vivo.

The mechanism of the enhanced antitumor activity of a combination of OGFand either gemcitabine or 5-FU needs to be defined. OGF is targeted tothe G₀/G₁ phase of the cell cycle and produces a notable delay inpancreatic cancer cell growth [41], but does not induce apoptosis [39].Gemcitabine and 5-FU are cytotoxic and induce programmed cell death [9,27, 30]. Therefore, the cytostatic action of OGF could be envisioned tochannel cells into the apoptotic pathway associated with gemcitabine or5-FU.

Gemcitabine is the standard of care for metastatic cancer [7, 13, 17,24, 42], and is in clinical trials as a single-agent chemotherapeuticfor locally advanced pancreatic cancer [1]. Treatment with gemcitabineis not curative for metastatic disease, and treatment with this agent asto its palliative benefit must be examined in the face of such factorsas toxicity [1, 17]. Given the urgent need for advancement in thetreatment of pancreatic cancer, combinations of drug therapies, many ofwhich involve a new agent plus gemcitabine, for pancreatic cancer havegained attention [5, 7, 17, 24]. The present report raises the excitingpotential of combining chemotherapy and biotherapy into a noveltreatment modality for human pancreatic cancer. OGF is not toxic, avoidsproblems related to drug resistance, has easy accessibility, and can beintegrated into the chronic use of chemotherapeutic agents. Moreover, itintroduces the possibility of using chemotherapeutic agents at lesstoxic concentrations and/or in chronic regimens (metronomicchemotherapy) [see 10, 16] in combination with a biotherapy. OGF used asa single-agent has been successful in a Phase I clinical trial withpatients with advanced unresectable pancreatic adenocarcinoma [33].During the chronic experiments in this study by Smith and colleagues[33], mean survival from the time of diagnosis was 8.7 to 9.5 months,depending on the route of drug administration, with some patients livingas long as 23 months. With the preclinical information in this reportshowing that a combination of OGF and gemcitabine has marked effects onpancreatic cancer in tissue culture and in xenografts, and the data fromthe Phase I clinical trial with OGF reporting a lack of toxicity andsuggesting efficacy, the prospect of clinical studies using combinationdrug therapy with OGF and gemcitabine appears to be warranted.

The observations in this study showing that the combination of OGF withgemcitabine has a potent inhibitory action on human pancreatic cancer,both in vitro and in vivo, are consistent with reports for OGF incombination with chemotherapy for treatment of squamous cell carcinomaof the head and neck (SCCHN) [14, 19]. Using tissue culture, McLaughlinand colleagues [19] demonstrated that OGF in combination with eitherpaclitaxel or carboplatin has a profound repressive influence on thegrowth of SCCHN. Jaglowski et al. [14] has reported that OGF incombination with paclitaxel markedly inhibited tumor growth inxenografts of SCCHN. In both in vitro and in vivo investigations, thecombination of OGF and chemotherapy was greater than that for theindividual compounds. In addition to pancreatic [38, 44] and SCCHN[19-21], OGF has been shown to influence the growth (in vitro and/or invivo) of a wide variety of cancers including neuroblastoma [22], renalcancer [2], and colon cancer [37]. These data indicate that combinedchemotherapy (e.g., gemcitabine, paclitaxel) and biotherapy (OGF) for avariety of cancers is likely.

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46. Zagon I S, Verderame M F, Allen S S, McLaughlin P J (2000) Cloning,sequencing, chromosomal location, and function of a cDNA encoding theopioid growth factor receptor (OGFr) in humans. Brain Res 856:75-83TABLE 6 Incidence and latency for tumor appearance of MIA PaCa-2pancreatic carcinoma cells in nude mice treated with OGF and/orgemcitabine (Gemzar). Parameter Control OGF Gemzar Gemzar/OGF N 10 10 1212 Tumor Incidence,  8/10  6/10  9/12 0/10^(a) day 10 Tumor Incidence,10/10 10/10 11/12 9/12 day 16 Latency to visible 10.1 ± 1.8 10.7 ± 0.811.1 ± 1.1 16.2 ± 1.2* tumor, d Latency to 13.2 ± 1.8 14.2 ± 0.8 13.1 ±1.0 19.5 ± 1.1* measurable tumor, dValues represent means ± SEM.^(a)Significantly different from every group by Chi-square analyses at p< 0.001.Significantly different from controls at p < 0.05(*) using ANOVA.

TABLE 7 Characteristics of nude mice 45 days after subcutaneousinoculation of MIA PaCa-2 pancreatic cancer cells and treatment with OGFand/or gemcitabine (Gemzar) Parameter Control OGF Gemzar Gemzar/OGF BodyWeight, g  33.3 ± 1.0  31.4 ± 1.6  27.4 ± 0.55  30.6 ± 0.7 Tumor Weight,g  5.5 ± 1.0  3.5 ± 0.5*  2.4 ± 0.1***  0.8 ± 0.1***++{circumflex over( )} Tumor Volume,mm³  8935 ± 1694  4849 ± 490***  3963 ± 123***  1477 ±53***+{circumflex over ( )} Spleen Weight, mg   761 ± 61   606 ± 121  454 ± 49*   437 ± 62*Data represent means ± SEM.Significantly different from controls at p < 0.05(*) and p < 0.001(***).Significantly different from OGF group at p < 0.05(+) and p < 0.01(++).Significantly different from the Gemzar-treated mice at p <0.05({circumflex over ( )}).

EXAMPLE 8

This study evaluated the effects of a combination of Opioid GrowthFactor (OGF) and paclitaxel on squamous cell carcinoma of the head andneck (SCCHN) using a tissue culture model of human SCCHN. Thecombination of OGF and paclitaxel was markedly inhibitory to SCCHNproliferation, reducing growth from control levels by 48% to 69% within48 hr. OGF in combination with carboplatin also depressed cell growth.The effect of a combination of OGF and paclitaxel or carboplatin onSCCHN growth was supra-additive, being greater than either of theindividual compounds. The action of OGF, but not paclitaxel, wasmediated by a naloxone-sensitive receptor and was completely reversible.OGF, but no other endogenous or exogenous opioid, altered replication ofSCCHN. OGF and paclitaxel depressed DNA synthesis, whereas onlypaclitaxel induced apoptosis. The combination of OGF and paclitaxel alsohad a supra-additive effect on the growth of another SCCHN, CAL-27,indicating the ubiquity of the combined drug activity. These datasuggest that the combination of a biotherapy (OGF) and chemotherapy(paclitaxel, carboplatin) may provide an enhanced antitumor action withrespect to SCCHN.

Given the promising nature of OGF (biotherapy), and of paclitaxel(chemotherapy), as antitumor agents in SCCHN, and the lack ofpreclinical data regarding the simultaneous use of OGF and paclitaxel,the present study was designed to explore the therapeutic potential of acombination of these modalities. Using a tissue culture model of theUM-SCC-1 cell line (SCC-1) derived from a well-differentiated recurrentsquamous cell carcinoma in the floor of the mouth, the effect ofconcomitant exposure to both OGF and paclitaxel were characterized ongrowth (e.g., reversibility, receptor mediation, specificity) andmechanism of action (apoptosis, necrosis, and cell proliferation).

Materials and Methods

Cell line and cell proliferation assays. The UM-SCC-1 cell line (SCC-1)was derived from a well-differentiated recurrent squamous cell carcinomain the floor of the mouth of a 73-yr old male (25). This cell line wasobtained from The University of Michigan, Cancer Research Laboratory(Thomas E. Carey, Ph.D., Director). CAL-27 human squamous cell carcinomacell line, derived from a poorly differentiated carcinoma of the tonguein a 56-yr old male (26), was obtained from the American Type CultureCollection (Manassas, Va.). Both cell lines were grown in Dulbecco's MEM(modified) media supplemented with 10% fetal calf serum, 1.2% sodiumbicarbonate, and antibiotics (5,000 Units/ml penicillin, 5 mg/mlstreptomycin, 10 mg/ml neomycin). The cell cultures were maintained in ahumidified atmosphere of 7% CO₂/93% air at 37° C.

Cells were seeded at equivalent amounts into either 6-well or 96-wellplates (Falcon) and counted 24 hr later to determine plating efficiency.OGF (10⁻⁶ M) and/or paclitaxel (10⁻⁸ M), or sterile water were addedbeginning 24 hr after seeding (=0 hr); media and compounds were replaceddaily. OGF was prepared in sterile water and paclitaxel was dissolved inDMSO at a concentration of 10⁻² M and further diluted into sterilewater; dilutions represent final concentrations of the compounds. Theconcentration of OGF that was utilized was selected based on previousevidence demonstrating growth inhibition of SCCHN (13); theconcentration of paclitaxel was selected from preliminary studies in ourlaboratory demonstrating that paclitaxel at 10⁻⁸ M, but not 10⁻⁷ M,inhibited cell growth but did not eliminate all cells over a 5-6 dayperiod of time (15).

Some experiments examined the effects of carboplatin and OGF. Dosages of10⁻⁷ M carboplatin and/or 10⁻⁶ M OGF were utilized. The dosage ofcarboplatin selected was based on previous reports (27, 28), as well aspreliminary studies in our laboratory.

Cell number was recorded either by using the MTS proliferation bioassay(Cell Titer 96 One Solution, Promega, Madison, Wis.) and measuringabsorbency after 4 hr on a Biorad (Model 3550) plate reader at 490 nmwith a 750 nm background absorbance screening, or by directly countingcells. The MTS assay utilized 10 wells/treatment. For manual counts,cells were harvested by trypsinization with 0.25% trypsin/0.53 mM EDTA,centrifuged, and counted with a hemacytometer. Cell viability wasdetermined by trypan blue staining. At least two aliquots per well, and4-10 wells/treatment, were counted at each time for manual counting.

For some experiments, the rate of growth over a 96-hr period of time wascalculated using linear regression analyses. The slopes of the lines(number of cells/hr) were compared by analysis of variance. Allcalculations were performed with GraphPad Prism software.

DNA synthesis, apoptosis and necrosis. To begin to determine themechanisms of action of paclitaxel and/or OGF, the effects of thesedrugs on DNA synthesis (BrdU incorporation), apoptosis (caspase-3activity), and necrosis (trypan blue positivity) were evaluated. Toexamine DNA synthesis, SCC-1 cells were seeded onto 22 mm diametercoverglasses placed in 6-well plates (3×10³ cells/coverglass). Cellswere treated with paclitaxel (10⁻⁸ M) and/or OGF (10⁻⁶ M) for 24 or 72hr; media and drugs were replaced daily. Three hours prior to fixingcells, 30 μM BrdU was added to cultures. At appropriate times, cellswere rinsed, fixed in 10% neutral buffered formalin, and stained withantibodies to BrdU (Roche, Indianapolis, Ind.). The number of positivecells was recorded using fluorescence microscopy. At least 1000cells/treatment at each time were counted.

Caspase-3-FITC positive staining was used to characterize early stagesof apoptosis (29). SCC-1 cells were seeded into 6-well plates andtreated with drugs beginning 24 hr later; drugs and media were replaceddaily. Cells were harvested after 1, 3 and 6 days of drug treatment, andprepared according to manufacturer's recommendations for FACS analysis(FACS cell sorter with a 15 mW argon ion laser at 488 nm; Becton,Dickinson and Company, Franklin Lakes, N.J.). For caspase-3identification, the APO-ACTIVE 3 antibody detection kit (CellTechnology, Mountain View, Calif.) was used. Three samples from eachtreatment were analyzed at each time point. The percent gated cellsrecorded by flow cytometry was considered caspase positive.

Chemicals. All chemicals and drugs were purchased from Sigma Chemicals(St. Louis, Mo.). Statistical analyses. Cell numbers and/or absorbencieswere analyzed using analysis of variance (one- or two-factor whereappropriate) (ANOVA) with subsequent comparisons made using Newman-Keulstests.

Results

Growth assays with paclitaxel and OGF. To establish the efficacy of thecombination of paclitaxel and OGF on growth of SCCHN, and to contrastthis with the effects of the individual drugs, growth curves of SCC-1cells were generated. Experiments on the growth of SCCHN were evaluatedby either cell counting or the MTS assay, and these methods werecomparable indicating that either technique was appropriate foranalysis. Growth curves are presented in FIG. 17A, and the rates ofgrowth obtained from the slopes of the growth curves (number ofcells/hr) are presented in FIG. 17B. After 48 hr of drug treatment, OGFor paclitaxel reduced cell number by approximately 10% (not significant)and 33% (p<0.01), respectively, from control levels. However, thecombination of OGF and paclitaxel reduced cell number by 48% (p<0.001)suggesting a synergistic effect of these drugs. At 72 and 96 hr inculture, OGF significantly reduced cell number from control values by10% and 23%, respectively, whereas paclitaxel reduced cell number by 25%and 51%, respectively. Exposure of SCC-1 cells for 72 and 96 hr to bothdrugs resulted in subnormal cell numbers, with a significant (p<0.001)decrease from control levels of 63% and 69%, respectively, beingrecorded. At 72 hr, the effect of both drugs (OGF and paclitaxel)displayed a synergistic effect on growth. Cells exposed to both OGF andpaclitaxel had markedly fewer cells than in comparison to culturesexposed to either the OGF or paclitaxel alone at 72 and 96 hr, and fromthe OGF group at 48 hr.

Rates of growth over the 4 day period of time were analyzed, and theresults demonstrated that cell growth for OGF and for paclitaxel werenotably reduced from control values (p<0.01) (FIG. 17B). Moreover, cellstreated with a combination of OGF and paclitaxel had a growth rate thatwas significantly decreased from both the OGF and paclitaxel groups atp<0.01 and p<0.05, respectively.

Growth assays with carboplatin and OGF. To inquire whether other agentsused in the treatment of SCCHN have a heightened response in combinationwith OGF, growth studies with carboplatin and OGF were performed (FIG.18). At 48, 72, and 96 hr of drug exposure, OGF reduced cell number by8-10% from control levels, whereas carboplatin reduced cell number at 72and 96 hr by 19% and 21%, respectively. The combination of OGF andcarboplatin reduced cell number relative to control values by 14-27% inthe 48-96 hr time period. Exposure of SCC-1 cells to both OGF andcarboplatin reduced cell number from the OGF group by approximately7-20% at 48-96 hr, and from the group treated with carboplatin alone byapproximately 14% and 12% at 48 and 72 hr, respectively.

Opioid receptor mediated effects of OGF and/or paclitaxel. In order todetermine whether the effects of OGF and/or paclitaxel were mediated byan opioid receptor, some cultures were exposed to naloxone (10⁻⁶ M), ashort-acting opioid antagonist. Cells were seeded into 96-well platesand treated with 10⁻⁶ M OGF, 10⁻⁶ M naloxone, 10⁻⁸ M paclitaxel, orcombinations at the same concentrations—OGF/naloxone,paclitaxel/naloxone, paclitaxel/OGF, and paclitaxel/OGF/naloxone.Individual plates were read at 24, 48, 72, and 96 hr after drugaddition. Relative to control levels, addition of OGF, paclitaxel,paclitaxel/OGF, and paclitaxel/OGF/naloxone inhibited cell growth from8.8% to 26.0% (FIG. 19). Addition of naloxone completely blocked thegrowth inhibitory effects of OGF alone, but had no effect on the growthinhibitory action of paclitaxel alone. Moreover, naloxone partiallyneutralized the enhanced inhibitory effect of the combination ofpaclitaxel and OGF; cell number of the paclitaxel/OGF/naloxone group wascomparable to cells exposed to paclitaxel, but was significantly reducedfrom control levels. Naloxone alone, at the concentration utilized, hadno effect on growth.

Reversibility of the inhibitory growth effects of OGF and/or paclitaxel.To establish whether the effect of OGF and/or paclitaxel on growth couldbe reversed by withdrawing cells from drug exposure, cultures of SCC-1cells were exposed for 48 hr to 10⁻⁶M OGF, 10⁻⁸ M paclitaxel, orpaclitaxel/OGF. After 2 days, half of the plates had media removed andfresh media added with no additional OGF or paclitaxel (i.e.,OGF-reversal; paclitaxel-reversal; paclitaxel/OGF-reversal groups); somecultures continued to receive new media and drugs. Within 48 hr, theOGF-reversal group had 16% more cells than the OGF group continuing withOGF exposure, however the paclitaxel-reversal group did not differ fromcells continuing to be treated with paclitaxel (FIGS. 20A, B). Cellcultures exposed to the combination of OGF and paclitaxel hadsignificantly fewer cells than cultures treated with OGF or paclitaxelalone, as well as the combination of these drugs withdrawn after 48 hr.The paclitaxel/OGF-reversal group did not differ from the paclitaxelalone or paclitaxel-reversal groups.

Specificity of opioid peptide(s) related to head and neck cancer cellgrowth. To determine whether other opioid peptides are related togrowth, SCC-1 cultures (1000 cells/well) were treated daily with 10⁻⁶ Mconcentrations of a variety of natural and synthetic opioid ligands(FIG. 21); in some cases, these ligands were specific for μ, δ, or κopioid receptors. Drugs included OGF, morphine, DAMGO, DPDPE(d-Pen,d-Pen-enkephalin), DADLE (d-Ala-D-Leu-enkephalin), dynorphin1-13, endomorphin-1, endomorphin-2, and β-endorphin. Except for OGF,which had a 19% decrease from control levels in absorbency readings,none of the drugs utilized had any inhibitory or stimulatory effect ongrowth.

Programmed cell death. No differences in necrosis could be observed inanalysis of the number of trypan blue positive cells in cultures orsupernatants of control cells and those treated with OGF and/orpaclitaxel. Examination of apoptosis was conducted by measurement ofcaspase-3 product (FIG. 22). Using flow cytometry, the percentages ofcaspase-3 positive cells after one day of treatment with OGF and/orpaclitaxel were negligible. However, within 3 days of exposure topaclitaxel or a combination of OGF and paclitaxel, there were 3.4- and5.6-fold more caspase positive cells than in control cultures. At 6 daysin culture, there were 12.7- and 13.9-fold more caspase reactive cellstreated with paclitaxel or OGF and paclitaxel, respectively, than incontrol cultures. No change from control levels in caspase reactivitycould be recorded in cells exposed to OGF on days 3 or 6.

BrdU incorporation into SCC-1 cells. BrdU labeling of SCCHN cells for 3hours and treatment for 24 hours with OGF, paclitaxel, or OGF andpaclitaxel showed a 31%, 24%, and 33%, respectively, decrease in thenumber of positive cells relative to controls (FIG. 23). After 3 days ofdrug treatment, the number of BrdU positive cells was decreased 61% fromcontrol levels in the OGF-treated cultures. The number of BrdU labeledcells was reduced 24% and 16% from control levels in the paclitaxel orpaclitaxel-OGF treated cultures, respectively.

Ubiquity of paclitaxel and OGF effects on growth of SCCHN. To determinethe ubiquity of the supra-additive effect of OGF and paclitaxel incontrast to either drug alone, the poorly-differentiatedSCCHN—CAL-27—was investigated (FIG. 24). Log-phase cultures of CAL-27were initially exposed to various concentrations of paclitaxel (10⁻⁷ Mto 10⁻¹⁰ M) in order to evaluate the sensitivity of these cells to thisagent. After 48 hr in culture (drug and media changed daily), treatmentwith paclitaxel at concentrations of 10⁻⁷, 10⁻⁸, 10⁻⁹, and 10⁻¹⁰ Mdepressed growth at 66%, 53%, 44%, and 22% from control levels. A dosageof 10⁻¹⁰ M paclitaxel was chosen for further study in order to examinethe magnitude of the combination of OGF and paclitaxel in the face of alower level of toxicity. After 48 hr, exposure of CAL-27 cells to eitherOGF (10⁻⁶ M), paclitaxel (10⁻¹⁰ M), or OGF (10⁻⁶ M) and paclitaxel(10⁻¹⁰ M) revealed 25%, 35%, and 61%, respectively, fewer cells than incontrol cultures. These differences in cell growth with exposure to OGFand/or paclitaxel differed significantly (p<0.001) from control levels,and the combination of OGF and paclitaxel differed from the OGF and thepaclitaxel treated cultures at p<0.001 and p<0.01, respectively.

Discussion

Data generated in this study demonstrate that the combination of OGF andpaclitaxel has a potent inhibitory effect on the growth of 2 cell linesof SCCHN in tissue culture. The antigrowth action of OGF and paclitaxelwas supra-additive, with the total inhibitory activity being greaterthan the sum of the parts (i.e., OGF or paclitaxel alone). Therepressive effects on growth of SCCHN observed with OGF and withpaclitaxel individually were consonant with previous results (e.g., 13,30, 31). The action of OGF on cell growth were mediated by analoxone-sensitive receptor. This naloxone-sensitive receptor ispresumed to be OGFr, because synthetic and natural opioids selective forclassical opioid receptors such as μ, δ, and κ did not influence cellreplication of SCC-1 as demonstrated in the present report and earlier(13, 22). OGF also was found to have a reversible action on thereplication of SCC-1, supporting the results from earlier studiesshowing that treatment with this compound does not lead to cytotoxicityand cell death (13, 21). On the other hand, the effects of paclitaxel onSCC-1 cells were neither blocked by naloxone nor could be reversed,indicating that the characteristics of this drug's action on SCC-1 ismarkedly different from that of OGF. Thus, this is the first report ofthe efficacy of using a combination of the biotherapeutic agent, OGF,and the chemotherapeutic agent, paclitaxel, to retard the growth ofSCCHN.

Although this report focused on the effects of OGF and paclitaxel onSCC-1 cells for detailed study, it is known that OGF, and paclitaxel,influence the growth of a variety of SCCHN cell lines (13, 32). Thepresent investigation demonstrates that not only does OGF and paclitaxelhave a supra-additive inhibitory effect on the SCC-1 cell number, but asimilar action can be found with another SCCHN cell line, CAL-27. Thus,the combination of OGF and paclitaxel appears to have more than asingular effect on one SCCHN line, and has a potent inhibitory action onthe growth of both well-differentiated (SCC-1) (25) andpoorly-differentiated (CAL-27) (26) SCCHN. Thus, it is reasonable toconclude that the effects of combination therapy with OGF and paclitaxelobserved herein also extend to other SCCHN cell lines.

Paclitaxel is a chemotherapeutic agent that prevents microtubuledepolymerization resulting in the arrest of proliferating cells in theG₂-M phase of the cell cycle and leading to cell death (33, 34).Additionally, paclitaxel modulates a number of intracellular eventswhich result in cellular apoptosis and ensuing nuclear degradation (35).OGF is known to not influence apoptosis (21), but is targeted to theG₀/G₁ phase of the cell cycle (17). Our experiments showed that SCCHNexposed to paclitaxel resulted in a marked increase in the number ofapoptotic cells within 3 days of initiation of drug treatment. By 6 daysof drug exposure, over one-half the SCCHN cells were apoptotic. OGF hadno effect on apoptosis of SCCHN, but produced a significant reduction inthe number of cells undergoing the S phase of DNA synthesis. Therefore,the mechanism for the enhancement by the combined effect of OGF andpaclitaxel as to growth inhibition could be related to delays in thecell cycle (the effect of OGF) which results in the recruitment of cellsinto the apoptotic pathway (the effect of paclitaxel).

To address the question whether OGF could be combined with agents otherthan taxols in order to treat SCCHN, a preliminary study was conductedwith the combination of OGF and a platinum analogue: carboplatin.Carboplatin causes a cross-linking of DNA strands by intercalation andthe creation of a bifunctional covalent link that in turn interrupts DNAsynthesis during the S phase of the cell cycle (36-38). This drug hasbeen shown to exhibit cytotoxicity through the induction of apoptosis(39, 40). The present results are the first to show that the effects ofa combination of carboplatin and OGF has potent inhibitory propertieswith respect to SCCHN. However, unlike the case for the taxanes and OGFwhich revealed a supra-additive action with OGF and paclitaxel, OGF andcarboplatin had an additive effect on growth. Presumably, these resultsindicate that OGF could be used in combination with more than one familyof chemotherapeutic agents (i.e., taxanes, platinums) to enhanceantitumor activity. Further studies are needed to characterize the modeof action of a combination of these two drugs. The end result may bethat OGF is a cytostatic drug, whereas paclitaxel and carboplatin induceprogrammed cell death, and that OGF contributes to cell death bychanneling cells into the apoptotic pathway.

Paclitaxel has been reported to be active in the treatment of squamouscell carcinoma of the head and neck, and Phase II evaluation has beensuccessful (6). Used as a single-agent therapy for SCCHN, this drugimproved response rate, as well as median survival time, in comparisonto cisplatin and 5-fluorouracil combination chemotherapy. However, 91%of the patients exposed to paclitaxel experienced neutropenia. AlthoughOGF has been approved in Phase I trials (41), OGF has not been usedclinically for the treatment of SCCHN. However, the efficacy of thiscompound has been demonstrated in xenograft experiments (14, 16). Thepresent report raises the exciting potential of combining chemotherapyand biotherapy into a novel treatment modality for SCCHN.

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EXAMPLE 9

The present report addresses the question of whether a combination ofOGF and paclitaxel influences growth of human SCCHN in vivo, and does sobeyond the efficacy of each compound. The effects of OGF and/orpaclitaxel on tumor incidence, appearance, size and metastasis, and onthe binding characteristics of the OGF receptor, were examined in axenograft model of SCCHN using human SCC-1 cells.

Material and Methods

Cell Lines

The UM-SCC-1 cell line (SCC-1) [8] was obtained from Cancer ResearchLaboratory at The University of Michigan (Dr. Thomas E. Carey,Director). Cells were grown in Dulbecco's MEM (modified) mediasupplemented with 10% fetal calf serum, 1.2% sodium bicarbonate, andantibiotics (5,000 Units/ml penicillin, 5 mg/ml streptomycin, 10 mg/mlneomycin). The cell cultures were maintained in a humidified atmosphereof 7% CO₂/93% air at 37° C. Cells were harvested by trypsinization with0.05% trypsin/0.53 mM EDTA, centrifuged, and counted with ahemacytometer. Cell viability was determined by trypan blue staining.

Animals and Tumor Cell Implantation

Male 4 week old nu/nu nude mice purchased from Harlan Laboratories(Indianapolis, Ind.) were housed in pathogen-free isolators in theDepartment of Comparative Medicine at the Penn State University Collegeof Medicine. All procedures were approved by the IACUC committee of thePenn State University College of Medicine and conformed to theguidelines established by NIH. Mice were allowed 48 hr to acclimateprior to beginning experimentation.

Tumor cells were inoculated into nude mice by subcutaneous injectioninto the right scapular region. Subcutaneous injections were performedwith at least 2×10⁶ cells per mouse; mice were not anesthetized for thisprocedure.

Chemotherapeutic Administration

Four groups of mice (n=12) were randomly assigned to receiveintraperitoneal injections of 10 mg/kg OGF daily, 8 mg/kg paclitaxelevery other day; 10 mg/kg OGF daily and 8 mg/kg paclitaxel every otherday, or 0.1 ml of sterile saline daily. In the group receiving combinedtherapy, OGF was injected prior to paclitaxel. Dosages were selectedbased on published reports [1, 17]. Paclitaxel was dissolved in DMSO andthen diluted in sterile saline; OGF was dissolved in sterile saline.Injections of drugs were initiated 1 hr after tumor cell inoculation.Preliminary studies were performed to determine whether DMSO alonealtered tumor response by injecting mice with 0.1 ml DMSO daily; nodifferences in tumor growth were found between injections of saline orDMSO thus data were combined for analyses. Mice were weighed weekly todetermine drug dosage.

Tumor Growth and Metastases

Mice were observed daily for the presence of tumors. The latency for avisible tumor to appear, and the time until tumors were measurable(i.e., 62.5 mm³), were recorded. Tumors were measured using calipersevery day. Tumor volume was calculated using the formula w²×1×π/6, wherethe length is the longest dimension, and width is the dimensionperpendicular to length [24].

Termination Day Measurements

According to institutional policies and IACUC guidelines, mice wereterminated when tumors became ulcerated, or tumors grew to 2 cm indiameter. Fifty 50 days following tumor cell inoculation andapproximately 35-40 days following initial tumor appearance, all micewere euthanized by an overdose of sodium pentobarbital (100 mg/kg) andkilled by cervical dislocation; mice (with tumors) were weighed. Tumorsand spleens were removed and weighed, and the lymph nodes, liver, andspleen examined for metastases.

Receptor Binding Analyses

Tumor tissues from some mice in each treatment group were removed at thetime of death, washed free of blood and connective tissue, andimmediately frozen in liquid nitrogen. Tissues were assayed followingthe procedures published previously [16]. Saturation binding isothermswere generated using GraphPad Prism software; binding affinity (K_(d))and capacity (B_(max)) values were provided by the computer software.

Plasma Levels of OGF

At the time of termination, trunk blood was collected from several micein each group. Plasma was separated and OGF levels were measured bystandard radioimmunoassay procedures using a kit from PeninsulaLaboratories (Belmont, Calif.). Plasma samples were assayed induplicate.

Statistical Analyses

Incidence of tumors was analyzed by chi-square tests. Latency for tumorappearance and tumor volume were analyzed using analysis of variance(ANOVA) with subsequent comparisons made using Newman-Keuls tests.Growth of tumors, termination day data (i.e., body weight, tumor weight,spleen weight), plasma levels of OGF, as well as binding capacity andaffinity of tumors, were compared by ANOVA and Newman-Keuls tests.

Survival data of the nude mice were analyzed using Kaplan-Meier plots.Tumor growth was analyzed using a non-linear mixed effects model forclustered data.

Results

SCC-1 Tumor Appearance and Growth

On day 13, when 75% of the mice in the saline-injected control group hadmeasurable tumors, 33% of the mice receiving OGF had a tumor; thesevalues differed significantly at p<0.05 (Table 8). Although fewer micein the paclitaxel and paclitaxel/OGF groups (66% and 70%, respectively)had measurable tumors compared to controls, these differences were notstatistically significant. On day 17 when 100% of the control mice hadmeasurable tumors, only 66% of the mice receiving OGF had tumors, and83% and 90% of the animals in the paclitaxel and paclitaxel/OGF groups,respectively, had tumors; however, no significant differences wererecorded (Table 9). All mice inoculated with SCC-1 cells developedtumors (Table 8), with 100% of the mice in the control group havingtumors by day 17 and every animal in the other groups having ameasurable tumor by day 28. The latency time for mice receiving OGF todevelop visible tumors was 11 days in comparison to controls that had amean latency of 7 days; this four-day delay was significantly differentat p<0.02 (Table 8). The mean latency time for visible tumors to appearwas comparable between mice in the control group and in the paclitaxeland paclitaxel/OGF groups. The mean latency time until tumors becamemeasurable ranged from 14 to 17 days, and did not differ between groups.

Changes in tumor volume over the 50 days of the experiment were analyzedusing a non-linear mixed effects model for clustered data (FIG. 25).These analyses compensated for the marked loss of paclitaxel micebeginning on day 20. Tumor volumes of mice in all 3 treatment groupswere significantly smaller than controls. Moreover, tumor volumes formice receiving combined therapy were significantly smaller than tumorsizes in groups receiving either treatment alone.

The weights of tumors on termination day (day 50) in the OGF and thepaclitaxel/OGF groups were reduced 29% and 62%, respectively, fromcontrol levels (Table 9). Evaluation of tumor volume on day 50 revealedthe OGF and paclitaxel/OGF groups had a reduction of 33% and 69%,respectively, from control values (Table 9). Because only one mouse inthe paclitaxel group was alive at this timepoint, analysis of tumorweight or volume were performed. Measurements of tumor weight and volumein the paclitaxel/OGF group on day 50 also revealed a decrease of 47%and 53%, respectively, from that occurring in the OGF group.

Survival

Survival curves for mice in each group are presented in FIG. 27. Two oftwelve mice receiving paclitaxel/OGF died within one week of initiationof the experiment; the cause(s) of these deaths appeared unrelated totumor development or the process of injection (e.g., ulceration). Micereceiving paclitaxel began dying within 20 days of treatment. By day 40,75% of the mice receiving paclitaxel had died, and at day 50 only onemouse in this group was alive. One mouse in the paclitaxel/OGF groupdied on day 42. No mouse in the OGF or control groups died during theexperimental period. Statistical comparisons of the survival curvesrevealed that death rates for paclitaxel mice were statisticallyreliable (p<0.0001) from all other groups. The average life span forpaclitaxel mice was 34.3±3.1 days in comparison to the 50-day life spanof other mice (day 50=termination day), and this difference wasstatistically significant from all 3 groups (p<0.001).

Body Weights and Gross Observations

Although all mice weighed approximately 22-23 g at the beginning of theexperiment (FIG. 26), mice receiving paclitaxel had a 10% reduction inbody weight at week 5 of the study and were subnormal by 9-10% on weeks6 and 7. On the termination date (i.e., day 50), mice receivingpaclitaxel weighed 28% less than control subjects, and weresignificantly less (p<0.001) in body weight than mice in the OGF andpaclitaxel/OGF groups (Table 9). No differences in body weights betweencontrol animals and those in the OGF or paclitaxel/OGF groups wererecorded.

Gross observations of the mice in the paclitaxel group revealeddistended abdomens, impacted bowel, and severe body weight loss.Pathological reports indicated colonic dilation and peritonitis; allother organ systems appeared normal. No pathological relevant findingscould be detected for mice in the control, OGF, or paclitaxel/OGFgroups.

Spleen weights did not differ among groups. In addition, no metastaseswere noted in the spleens, liver, or axillary lymph nodes of mice in anygroup.

OGFr Binding Characteristics

Specific and saturable binding for OGFr, with a one-site model ofbinding, was recorded in tumors collected from all 4 groups of mice.Tumors from the paclitaxel group were obtained at days 47 to 50, whereasspecimens from all other groups were harvested on the final day ofexperimentation (day 50). Binding affinity (K_(d)) for OGF to OGFrranged from 1.0 to 2.1 nM and did not differ among groups (Table 10).However, values for binding capacity (B_(max)) were almost 2-fold higherin the OGF and paclitaxel group relative to control subjects (˜15fmol/mg protein) (Table 10).

Plasma Levels of OGF

OGF levels in the plasma of nude mice bearing SCC-1 tumors ranged from282 to 617 pg/ml. No differences were noted between control mice withtumors and those treated with OGF, paclitaxel, or paclitaxel/OGF.

Discussion

The present results show that a combination of OGF and paclitaxel has apotent inhibitory effect on the growth of SCC-1 in nude mice, awell-differentiated human tumor model of SCCHN. The antigrowth action ofOGF and paclitaxel was synergistic, with the total inhibitory activitybeing greater than the sum of the parts (i.e., OGF or paclitaxel alone).This supra-additive effect of OGF and paclitaxel was most evident inmeasurements of tumor weight and volume. These results performed underin vivo conditions extend earlier observations conducted in tissueculture [13] in which a combination of OGF and paclitaxel had asynergistic repressive effect on cell number. Thus, this is the firstreport of the efficacy of using a combination of the biotherapeuticagent, OGF, and the chemotherapeutic agent, paclitaxel, to retard thegrowth of SCCHN in vivo. Although this study focused on one SCCHN cellmodel, SCC-1, it is known that OGF, and paclitaxel, influence the growthof a variety of SCCHN cell lines [10, 14]. Therefore, it is reasonableto conclude that the effects of combination therapy with OGF andpaclitaxel observed herein also extend to other SCCHN cell lines.

An important observation recorded in the present investigation was thewell-known [7, 28] marked systemic toxicity from paclitaxel which wasmanifested in significant reductions in body weight and survival, aswell as gross lesions and pathological signs, and the attenuation ofthis toxicity by simultaneous administration of OGF. However, theamelioration of paclitaxel toxicity by OGF was not accompanied by adiminution in the antitumor action of paclitaxel. In fact, thecombination of OGF and paclitaxel had an effect on tumor growth (i.e.,weight, volume) that exceeded paclitaxel alone (or OGF alone). Theseresults would suggest that chemotherapeutic levels of paclitaxel werebetter tolerated and compatible with survival when given concomitantlywith the biotherapeutic agent, OGF. The alleviation of toxicity of oneagent by administration of another drug is not without precedence [3,9]. In and by itself, the finding of protection afforded by OGF from theside effects produced by taxanes is important. However, the combinationof OGF and paclitaxel could allow even higher cytostatic doses ofpaclitaxel to be administered in order to improve the therapeuticefficacy of this agent. Indeed, the success of chemotherapeutic agentsis often limited by an intrinsic resistance of the cancer cells, and theavailability of increasing the concentration of drugs like paclitaxelwithout an accompanying increase in toxicity would be advantageous.Finally, it is unclear as to whether the effectiveness of a combinationof OGF and paclitaxel is animal specific and/or is due to the lack ofimmune components in nude mice. Because myelosuppression is a main sideeffect of chemotherapy it would be valuable to explore the immunologicalramifications of OGF/paclitaxel therapy in the understanding of drugmechanism.

Previous studies have shown that surgical specimens of SCCHN havesignificantly fewer OGF receptors than normal mucosa [18].Translation/posttranslation of OGFr protein rather than irregularitiesin OGFr gene transcription may be involved in this decrease in receptornumber. These authors postulate that the number of OGF receptors may bedependent on tumor size, and that the progressive diminishment in OGFreceptors in SCCHN compromises the inhibitory activity of OGF andthereby contributes to an accelerated cell proliferation. In the presentinvestigation, tumor tissue from animals treated with OGF or paclitaxeland inoculated with SCCHN had over a 2-fold greater binding capacitythan neoplastic tissue from control subjects. And, although notstatistically significant, even those animals receiving a combination ofpaclitaxel and OGF had an increase of 38% in binding capacity. If thehypothesis put forth by McLaughlin and colleagues [17] is correct, itwould be understandable that the smaller SCCHN tumors in OGF and/orpaclitaxel mice would have more OGF receptors (and grow slower) thanthose in control mice because of the repressed cell replication and lessimpaired OGF-OGFr axis.

Paclitaxel is a chemotherapeutic agent that prevents microtubuledepolymerization resulting in the arrest of proliferating cells in theG₂-M phase of the cell cycle which leads to cell death [31, 32].Additionally, paclitaxel modulates a number of intracellular eventswhich result in cellular apoptosis and ensuing nuclear degradation [27].OGF does not influence apoptosis [31], but is targeted to the G₀/G₁phase of the cell cycle [32]. Earlier experiments in tissue cultureshowed that SCCHN exposed to paclitaxel resulted in a marked increase inthe number of apoptotic cells. Therefore, the mechanism for the enhancedgrowth inhibition in vivo by the combined effect of OGF and paclitaxelcould be related to delays in the cell cycle (the effect of OGF) whichresults in the recruitment of cells into the apoptotic pathway (theeffect of paclitaxel).

Paclitaxel has been reported to be active in the treatment of squamouscell carcinoma of the head and neck, and Phase II evaluation has beensuccessful [4]. Used as a single-agent therapy for SCCHN, this drugimproved response rate, as well as median survival time, in comparisonto cisplatin and 5-fluorouracil combination chemotherapy. However, 91%of the patients exposed to paclitaxel experienced neutropenia. AlthoughOGF has been approved in Phase I trials [26], OGF has not been usedclinically for the treatment of SCCHN. However, the efficacy of thiscompound for treatment of SCCHN has been demonstrated in xenograftexperiments [16, 17]. The present report raises the exciting potentialof combining chemotherapy and biotherapy into a novel treatment modalityfor SCCHN. With the preclinical information that a combination of OGFand paclitaxel has a synergistic effect on SCCHN in xenografts, theprospect of clinical studies should be considered.

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35. Zagon I S, Wu Y, McLaughlin P J (1999) Opioid growth factor andorgan development in rat and human embryos. Brain Res 839:313-322 TABLE8 Incidence and latency for tumor appearance of SCC-1 squamous cellcarcinoma cells in nude mice treated with OGF and/or paclitaxel.Parameter Control OGF Paclitaxel Paclitaxel/OGF N 12 12 12 10 Incidenceof  9/12 4/12^(a)  8/12 7/10 measurable tumor (day 13) Incidence of12/12 8/12 10/12 9/10 measurable tumor (day 17) Latency to  7.2 ± 0.511.2 ± 1.5^(b)  7.4 ± 1.4  8.6 ± 0.8 visible tumor (days) Latency to14.2 ± 0.6 17.0 ± 1.5 14.8 ± 1.7 15.5 ± 1.5 measurable tumor (days)Values represent means ± SEM.^(a)Significantly different from the control group by Chi-squareanalyses at p < 0.05.^(b)Significantly different at p < 0.02 from controls using ANOVA.

TABLE 9 Characteristics of nude mice 50 days after subcutaneousinoculation of SCC-1 squamous carcinoma cells and treatment (i.p.) withOGF and/or paclitaxel Parameter Control OGF Paclitaxel Paclitaxel/OGFBody Weight, g 31.6 ± 0.7   32.0 ± 0.5 22.6 ± 0.8***+++{circumflex over( )}{circumflex over ( )}{circumflex over ( )}  31.8 ± 1.1 Tumor Weight,g 2.4 ± 0.2  1.7 ± 0.2** N.A.  0.9 ± 0.7***+++ Tumor Volume, mm³ 3896 ±535   2590 ± 364* N.A.  1223 ± 238***+ Spleen Weight, mg 243 ± 25    225± 12  243 ± 8   197 ± 19 Metastases none none none noneData represent means ± SEM.N.A. = data not available because only one mouse was alive on day 50;spleen and body weights for the paclitaxel group only were calculated onthe day each mouse died.Significantly different from controls at p < 0.05(*), p < 0.01(**) and p< 0.001(***).Significantly different from OGF group at p < 0.05(+) and p <0.001(+++).Significantly different from the paclitaxel-treated mice at p <0.001({circumflex over ( )}{circumflex over ( )}{circumflex over ( )}).

TABLE 10 Receptor binding analysis of OGFr in SCC-1 tumors from micetreated with OGF and/or paclitaxel. Parameter Control OGF PaclitaxelPaclitaxel/OGF K_(d), nM  1.0 ± 0.1  2.1 ± 0.3  1.2 ± 0.2  1.4 ± 0.3B_(max), 14.9 ± 1.2 27.2 ± 2.2* 27.8 ± 1.6* 20.5 ± 2.1 fmol/mg proteinData represent means ± SEM.Significantly different from controls at p < 0.05(*).

It should be understood that the embodiments described herein are forillustrative purposes only and that various modifications or changes inlight thereof will be suggested to persons skilled in the art and are tobe included within the spirit and purview of this application.

1. A pharmaceutical composition for treating neoplasias in an animal orhuman which are characterized by an opioid growth factor receptor,comprising: therapeutically effective amounts of at least onechemotherapeutic, biotherapeutic, and/or radionuclide agent with opioidgrowth factor; and a carrier.
 2. The pharmaceutical composition of claim1 wherein said agent is a therapeutic agent.
 3. The pharmaceuticalcomposition of claim 2, wherein the neoplasias includes not limited topancreatic cancer, squamous cell cancer of the head and neck, breastcancer, colorectal cancer, renal cancer, brain cancer, prostate cancer,bladder cancer, bone or joint cancer, uterine cancer, cervical cancer,endometrial cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin'slymphoma, melanoma, leukemias, lung cancer, ovarian cancer,gastrointestinal cancer, Kaposi's sarcoma, liver cancer, pharyngealcancer and laryngeal cancer.
 4. The pharmaceutical composition of claim3, wherein the chemotherapeutic agent is selected from but not limitedto the group comprising: busulfan, cisplatin, carboplatin, chlorambucil,cyclophosphamide, ifosfamide, dacarbazine (DTIC), mechlorethamine(nitrogen mustard), melphalan carmustine (BCNU) lomustine (CCNU), 5-FU,capecitabine, methotrexate, gemcitabine, cytarabine (ara-C), fludarabinedactinomycin, daunorubicin, doxorubicin (Adriamycin), idarubicin,mitoxantrone, paclitaxel, docetaxel, etoposide (VP-16), vinblastine,vincristine, vinorelbine prednisone, dexamethasone, tamoxifen,fulvestrant, anastrozole, letrozole, megestrol acetate, bicalutamide,flutamide, leuprolide, goserelin, L-asparaginase, and tretinoin,gemcitabine, paclitaxel, carboplatin, and 5-FU.
 5. The pharmaceuticalcomposition of claim 4, wherein the therapeutic effective amount of OGFis about 100 to 400 μg/kg body weight when administered intravenously.6. The pharmaceutical composition of claim 5, wherein the therapeuticeffective amount of the chemotherapeutic agent ranges from about 100 to1000 mg/m² when administered intravenously.
 7. A method for treatingneoplasias characterized by an opioid growth factor receptor in ananimal or human in need of such treatment, comprising: administering tosaid animal or human therapeutically effective amounts of each of atleast one neoplasia-treating agent and opioid growth factor.
 8. Themethod of claim 7 wherein said neoplasia treating agent is radiation. 9.The method of claim 7 wherein said neoplasia treating agent is abiotherapy agent.
 10. The method of claim 7 wherein said neoplasiatreating agent is a chemotherapy agent.
 11. The method of claim 7wherein said neoplasia treating agent is a radionuclide.
 12. The methodof claim 7, wherein the opioid growth factor specifically binds to anopioid growth factor receptor.
 13. The method of claim 7, wherein theneoplasias includes but is not limited to pancreatic cancer, squamouscell cancer of the head and neck, breast cancer, colorectal cancer,renal cancer, brain cancer, prostate cancer, bladder cancer, bone orjoint cancer, uterine cancer, cervical cancer, endometrial cancer,multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma, melanoma,leukemias, lung cancer, ovarian cancer, gastrointestinal cancer,Kaposi's sarcoma, liver cancer, pharyngeal cancer and laryngeal cancer.14. The method of claim 7, wherein the neoplasia-treating agent is achemotherapeutic agent including but is not limited to gemcitabine,paclitaxel, carboplatin, and 5-fluorouracil.
 15. The method of claim 7,wherein the therapeutic effective amount of opioid growth factoradministered is about 100 to 400 μg/kg body weight per day whenadministered intravenously.
 16. The method of claim 7, furthercomprising: administering a chemotherapeutic agent sequentially orsimultaneously with opioid growth factor in therapeutically effectiveamounts ranging from about 100 to 1000 mg/M² intravenously (OGFcontinuous treatment over a period of between about 10 to 60 minutes atleast once a week for about three to ten weeks followed by a one tothree week rest period; administering the chemotherapeutic agent atleast once weekly for about one to five weeks after the one to threeweek rest period; and repeating the administration of thechemotherapeutic agent every two to eight weeks.
 17. The methodaccording to claim 12, wherein opioid growth factor is givencontinuously throughout the treatment period, when the therapeuticagent(s) is/are administered intermittently.
 18. The method of claim 10,wherein the route of administration of the at least one chemotherapeuticagent and opioid growth factor is selected from the group consisting ofparenterally, including intravenously, intramuscularly orintraperitoneally; subcutaneously, implanted osmotic pump andtransdermal patch.
 19. A method of decreasing the toxicity of paclitaxelcomprising: administering with said paclitaxel an effective amount ofopioid growth factor.
 20. A method of increasing the anti-neoplasticeffects of a chemotherapeutic agent selected from the group includingconsisting of: gemcitabine, paclitaxel, carboplatin, and 5-FUcomprising: introducing to said neoplastic cells, in combination withsaid chemotherapeutic agent, a therapeutically effective amount of OGF,wherein the neoplastic cell killing observed with said combination isgreater than the neoplastic cell killing of the chemotherapeutic agentalone.
 21. A method for killing neoplastic cells characterized by anopioid growth factor receptor in an animal or human in need of suchtreatment, comprising: administering to said animal or human atherapeutically effective amounts of a chemotherapeutic agent selectedfrom and not limited to the group comprising: gemcitabine, paclitaxel,carboplatin, and 5-FU; and opioid growth factor.
 22. A method forkilling neoplastic pancreatic cells characterized by an opioid growthfactor receptor in an animal or human in need of such treatment,comprising: administering to said animal or human a therapeuticallyeffective amounts of a chemotherapeutic agent selected from but notlimited to the group compromising: gemcitabine, and 5-FU; and opioidgrowth factor.
 23. A method for killing neoplastic squamous cellscharacterized by an opioid growth factor receptor in an animal or humanin need of such treatment, comprising: administering to said animal orhuman a therapeutically effective amount of a chemotherapeutic agentselected from the group consisting of paclitaxel, and carboplatin; andopioid growth factor.
 24. A pharmaceutical composition for treatingneoplasias in an animal or human which are characterized by an opioidgrowth factor receptor, comprising: therapeutically effective amounts ofat least one therapeutic agent selected from but not limited to thegroup comprising of a mitotic inhibitor, an anti-metabolite, and analkylating agent; and opioid growth factor; and a carrier.
 25. Thepharmaceutical composition of claim 23 wherein said chemotherapeuticagent is selected from the group comprising but not limited to:busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide,ifosfamide, dacarbazine (DTIC), mechlorethamine (nitrogen mustard), andmelphalan. 5-fluorouracil, capecitabine, methotrexate, gemcitabine,cytarabine (ara-C), and fludarabine, paclitaxel, docetaxel, etoposide(VP-16), vinblastine, vincristine, and vinorelbine.